Mutant sodium channel nav 1.7 and methods related thereto

ABSTRACT

Described are mutant Na v 1.7 sodium channel alpha-subunits and nucleic acid sequences encoding such mutants. Further described are methods for characterizing a nucleic acid sequence that encodes a Na v 1 sodium channel alpha-subunit, methods for determining a Na v 1.7 haplotype, methods for determining a subject&#39;s predisposition to a neurologic disorder associated with a sodium channel mutation, and methods of identifying a compound that modulates mutant Na v 1.7 sodium channels. Other materials, compositions, articles, devices, and methods relating to mutant Na v 1.7 sodium channels are also described herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of copending application Ser. No.10/585,717, filed Jul. 11, 2006, which was the National Stage ofInternational Application No. PCT/US2005/002059, filed Jan. 21, 2005,which claims benefit of U.S. Provisional Application No. 60/538,149,filed Jan. 21, 2004. application Ser. No. 10/585,717, filed Jul. 11,2006, International Application No. PCT/US2005/002059, filed Jan. 21,2005, and U.S. Provisional Application No. 60/538,149, filed Jan. 21,2004, are hereby incorporated herein by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under federal grantR01-NS-32666 awarded by the National Institutes of Health. TheGovernment has certain rights to this invention.

BACKGROUND

Voltage-gated sodium channels are transmembrane proteins that mediateregenerative inward currents that are responsible for the initialdepolarization of action potentials in excitable cells, such as neuronsand muscle. Sodium channels are typically a complex of various subunits,the principle one being the alpha-subunit. The alpha-subunit is thepore-forming subunit, and it alone is sufficient for all known sodiumchannel function. However, in certain sodium channels, smaller,auxiliary subunits called beta-subunits are known to associate with thelarger alpha-subunit and are believed to modulate some of the functionsof the alpha-subunit. (See Kraner, et al. (1985) J Biol Chem260:6341-6347; Tanaka, et al. (1983) J Biol Chem 258:7519-7526;Hartshorne, et al. (1984) J Biol Chem 259:1667-1675; Catterall, (1992)Physiol Rev 72:S14-S48; Anderson, et al. (1992) Physiol Rev72:S89-S158.) A review of sodium channels is presented in Catterall,(1995) Ann Rev Biochem 64:493-531.

The primary structures of sodium channel alpha-subunits from a varietyof tissues (brain, peripheral nerve, skeletal muscle, and cardiacmuscle) and organisms (jellyfish, squid, eel, rat, human) have beenidentified, and their amino acid sequences show individual regions whichhave been conserved over a long evolutionary period (see Alberts, etal., eds., “Molecular Biology of the Cell” 534-535, Garland Pub., NewYork, N.Y. (1994)). From these studies it is known that thealpha-subunit of a sodium channel is a large glycoprotein containingfour homologous domains (labeled I-IV in FIG. 1) connected byintracellular loops. The N-terminus of the alpha-subunit extendsintracellularly at domain I (i.e., DI) and the C-terminus of thealpha-subunit extends intracellularly at domain IV (i.e., DIV). In theplasma membrane, the four domains orient in such a way as to create acentral pore whose structural constituents determine the selectivity andconductance properties of the sodium channel.

Each domain of the sodium channel alpha-subunit contains sixtransmembrane alpha-helices or segments (labeled 1-6 in FIG. 1). Five ofthese transmembrane segments are hydrophobic, whereas one segment ispositively charged with several lysine or arginine residues. This highlycharged segment is the fourth transmembrane segment in each domain.Extracellular loops connect segment 1 (i.e., S1) to segment 2 (i.e., S2)and segment 3 (i.e., S3) to segment 4 (i.e., S4). Intracellular loopsconnect S2 to S3 and S4 to segment 5 (i.e., S5). An extracellularre-enterant loop connects S5 to segment 6 (i.e., S6). (See Agnew, et al.(1978) Proc Natl Acad Sci USA 75:2606-2610; Agnew, et al. (1980) BiochemBiophys Res Comm 92:860-866; Catterall, (1986) Ann Rev Biochem55:953-985; Catterall, (1992) Physiol Rev 72:S14-S48.)

Voltage-gated sodium channels can be named according to a standardizedform of nomenclature outlined in Goldin, et al. (2000) Neuron28:365-368. According to that system, voltage-gated sodium channels aregrouped into one family from which nine mammalian isoforms and have beenidentified and expressed. These nine isoforms are given the namesNa_(v)1.1 through Na_(v)1.9. Also, splice variants of the variousisoforms are distinguished by the use of lower case letters followingthe numbers (e.g., “Na_(v)1.1a”).

Because of the important role sodium channels play in the transmissionof action potentials in excitable cells like neurons and muscle, sodiumchannels have been implicated in many sensory, motor, and neurologicdisorders. Accordingly, sodium channels have been the focus of muchscientific research. However, while a great deal has been learned aboutsodium channels, there remains a need for further understanding of thefunctioning of sodium channels, and means to diagnose, predict, prevent,and treat diseases, disorders, and conditions that result fromvariations and abnormalities of sodium channels. These and other objectsand advantages of the materials, compositions, articles, devices, andmethods described herein, as well as additional inventive features, willbe apparent from the following disclosure.

BRIEF SUMMARY

In accordance with the purposes of the disclosed materials,compositions, articles, devices, and methods, as embodied and broadlydescribed herein, the disclosed subject matter, in one aspect, relatesto a method of characterizing a nucleic acid sequence that encodes aNa_(v)1.7 sodium channel alpha-subunit, wherein the method comprises thestep of identifying mutations at one or more sites in regions of thenucleic acid sequence that encode an intracellular N-terminal region, anextracellular loop in domain I, an intracellular loop between domains Iand II, an intracellular loop between domains II and III, anintramembrane region of domain II, or any combination thereof, suchidentified nucleotides indicating the character of the nucleic acidsequence.

In another aspect, the disclosed subject matter relates to a method fordetermining a Na_(v)1.7 haplotype in a human subject, wherein the methodcomprises identifying one or more nucleotides encoding amino acidresidues 62, 149, 641, 655, 739, 1123, or any combination thereof,wherein the nucleotide or nucleotides indicate the haplotype.

In yet another aspect, the disclosed subject matter relates to a methodfor determining a subject's predisposition to a neurologic disorderassociated with a sodium channel mutation comprising comparing thesubject's Na_(v)1.7 haplotype with one or more reference haplotypes thatcorrelate with the neurologic disorder, a similar haplotype in thesubject's Na_(v)1.7 haplotype as compared to the reference haplotype orhaplotypes indicating a predisposition to the neurologic disorder.

In a still further aspect, described herein is a method of identifying acompound that modulates mutant Na_(v)1.7 sodium channels, wherein themethod comprises contacting with a test compound a cell containing amutant Na_(v)1.7 nucleic acid that encodes a mutant Na_(v)1.7 sodiumchannel comprising one or more mutations at residue 62, residue 149,residue 641, residue 655, residue 739, or residue 1123, detectingNa_(v)1.7 sodium channel activity, and comparing the Na_(v)1.7 sodiumchannel activity in the contacted cell with the amount of Na_(v)1.7sodium channel activity in a control cell, wherein the control cell isnot contacted by the test compound, an increased or decreased Na_(v)1.7sodium channel activity in the test cell as compared to the control cellindicating a compound that modulates mutant Na_(v)1.7 sodium channels.

Also, described herein are isolated nucleic acids comprising nucleotidesequences encoding mutant Na_(v)1.7 sodium channel alpha-subunits,expression vectors made from such nucleic acids, cultured cellscomprising such vectors, and methods of making mutant Na_(v)1.7 sodiumchannel alpha-subunits comprising culturing such cells under conditionsallowing expression of the polypeptide encoded by the nucleic acids,wherein the polypeptide comprises a mutant Na_(v)1.7 sodium channelalpha-subunit. Further, described herein are isolated polypeptidescomprising mutant Na_(v)1.7 sodium channel alpha-subunits and fragmentsthereof as well as purified antibodies that bind to epitopes of suchmutant Na_(v)1.7 sodium channel alpha-subunits.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is a diagram of the secondary structure of a sodium channelalpha-subunit. Not shown is the pore region in each of the four domains,which consists of an inward loop between transmembrane regions 5 and 6.

FIG. 2 is a diagram showing the segregation of the N641Y mutation andphenotypic findings of kindred 4425. The following abbreviations areused in the diagram: “fs” means febrile seizures; “afs” means afebrileseizures; “+” means wild type; and “m” means mutant.

FIG. 3 is a diagram of the secondary structure of a Na_(v)1.7 sodiumchannel alpha-subunit where the locations of various mutations areidentified.

FIG. 4 is a graph showing current voltage relationships of whole-cellcurrents. Full-length wild-type SCN9A and mutant SCN9A (K655R and N641Y)constructs were transiently transfected into tsA201 cells. Currents wereelicted by test pulses from −60 mV to +40 mV in 5 mV increments. Atnegative potentials, K655R has a higher current density than wild type.At positive potentials, N641Y has reduced current density compared towild-type, p<0.05.

DETAILED DESCRIPTION

The materials, compositions, articles, devices, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subjectmatter, and methods and the Examples included therein and to the Figuresand their previous and following description.

Before the present materials, compositions, articles, devices, andmethods are disclosed and described, it is to be understood that theaspects described below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Disclosed herein are materials, compositions, and components that can beused for, can be used in conjunction with, can be used in preparationfor, or are products of the disclosed method and compositions. These andother materials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a Na_(v)1.7 sodium channel is disclosed and anumber of modifications that can be made to a number of amino acidresidues or nucleotides, including those related to the mutant Na_(v)1.7sodium channel are discussed, each and every combination and permutationthat are possible are specifically contemplated unless specificallyindicated to the contrary. Thus, if a class of substituents A, B, and Care disclosed as well as a class of substituents D, E, and F and anexample of a combination molecule, A-D is disclosed, then even if eachis not individually recited, each is individually and collectivelycontemplated. Thus, in this example, each of the combinations A-E, A-F,B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated andshould be considered disclosed from disclosure of A, B, and C; D, E, andF; and the example combination A-D. Likewise, any subset or combinationof these is also specifically contemplated and disclosed. Thus, forexample, the sub-group of A-E, B-F, and C-E are specificallycontemplated and should be considered disclosed from disclosure of A, B,and C; D, E, and F; and the example combination A-D. This conceptapplies to all aspects of this disclosure including, but not limited to,steps in methods of making and using the disclosed compositions. Thus,if there are a variety of additional steps that can be performed it isunderstood that each of these additional steps can be performed with anyspecific embodiment or combination of embodiments of the disclosedmethods, and that each such combination is specifically contemplated andshould be considered disclosed.

Throughout this specification, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings^(.)

As used in the specification and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a nucleotide”includes mixtures of two or more such nucleotides, reference to “anamino acid” includes mixtures of two or more such amino acids, referenceto “the sodium channel” includes mixtures of two or more such sodiumchannels, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “the array can optionally comprise themost commonly found allele at a second . . . position” means that themost commonly found allele at a second position may or may not bepresent in the array and that the description includes both arrayswithout the most commonly found allele at the second position and arrayswhere there is the most commonly found allele at the second position.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint.

“Subject,” as used herein, means an individual. In one aspect, thesubject is a mammal such as a primate, and, in another aspect, thesubject is a human. The term “subject” also includes domesticatedanimals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs,sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat,guinea pig, etc.).

“Na_(v)1.7,” as used herein, refers to an isoform of a sodium channelknown in the art by names such as NaS, hNE-Na, and PN1. The traditionalgene symbol for a Nav1.7 sodium channel is SCN9A, and thus the termNa_(v)1.7, as used herein, is synonymous with the term SCN9A. There area variety of sequences related to the Na_(v)1.7 gene having thefollowing Genbank Accession Numbers: NM 002977 (human), U35238 (rabbit),X82835 (human), U79568 (rat), and AF000368 (rat), these nucleic acidsequences, the polypeptides encoded by them, and other nucleic acid andpolypeptide sequences are herein incorporated by reference in theirentireties as well as for individual subsequences contained therein.

There are a variety of compositions disclosed herein that are amino acidbased, including for example Na_(v)1.7 sodium channel alpha-subunits.Thus, as used herein, “amino acid,” means the typically encounteredtwenty amino acids which make up polypeptides. In addition, it furtherincludes less typical constituents which are both naturally occurring,such as, but not limited to formylmethionine and selenocysteine, analogsof typically found amino acids, and mimetics of amino acids or aminoacid functionalities. Non-limiting examples of these and other moleculesare discussed herein.

As used herein, the terms “peptide” and “polypeptide” refer to a classof compounds composed of amino acids chemically bound together.Non-limiting examples of these and other molecules are discussed herein.In general, the amino acids are chemically bound together via amidelinkages (CONH); however, the amino acids may be bound together by otherchemical bonds known in the art. For example, the amino acids may bebound by amine linkages. Peptide as used herein includes oligomers ofamino acids and small and large peptides, including polypeptides andproteins.

There are a variety of compositions disclosed herein that are nucleicacid based, including for example the nucleic acids that encode, forexample, Na_(v)1.7 sodium channel alpha-subunits. Thus, as used herein,“nucleic acid” means a molecule made up of, for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. A nucleic acid can bedouble stranded or single stranded. It is understood that, for example,when a vector is expressed in a cell the expressed mRNA will typicallybe made up of A, C, G, and U. Likewise, it is understood that if, forexample, an antisense molecule is introduced into a cell or cellenvironment through, for example, exogenous delivery, it is advantageousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

As used herein, “nucleotide” is a molecule that contains a base moiety,a sugar moiety and a phosphate moiety. Nucleotides can be linkedtogether through their phosphate moieties and sugar moieties creating aninternucleoside linkage. The base moiety of a nucleotide can beadenine-9-yl (A), cytosine-1-yl (C), guanine-9-yl (G), uracil-1-yl (U),and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or adeoxyribose. The phosphate moiety of a nucleotide is pentavalentphosphate. A non-limiting example of a nucleotide would be 3′-AMP(3′-adenosine monophosphate) or 5′-GMP (5′-guanosine monophosphate).

“Nucleotide analog,” as used herein, is a nucleotide which contains sometype of modification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

“Nucleotide substitutes,” as used herein, are molecules having similarfunctional properties to nucleotides, but which do not contain aphosphate moiety, such as peptide nucleic acid (PNA). Nucleotidesubstitutes are molecules that will recognize nucleic acids in aWatson-Crick or Hoogsteen manner, but which are linked together througha moiety other than a phosphate moiety. Nucleotide substitutes are ableto conform to a double helix type structure when interacting with theappropriate target nucleic acid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger, et al. (1989) ProcNatl Acad Sci USA, 86:6553-6556.)

A “Watson-Crick interaction” is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A “Hoogsteen interaction” is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH₂ or O) at the C6 position of purinenucleotides.

“Deletion,” as used herein, refers to a change in an amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent relative to the reference sequence.

“Insertion” or “addition,” as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thereference sequence.

“Substitution,” as used herein, refers to the replacement of one or moreamino acids or nucleotides by one or more different amino acids ornucleotides, respectively, in a reference sequence.

“Isolated,” as used herein refers to material, such as a nucleic acid ora polypeptide, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with it as found in itsnaturally occurring environment. Although, the isolated materialoptionally comprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a locus in the cell(e.g., genome or subcellular organelle) not native to a material foundin that environment. The alteration to yield the synthetic material canbe performed on the material within or removed from its natural state.

Characterizing Mutant Na_(v)1.7 Nucleic Acid Sequences

It has been found that, in certain neurologic disorders, specific sitesin the Na_(v)1.7 gene are mutated, i.e., the nucleotide at a specificposition or at specific positions differs from that observed in the mostcommonly found Na_(v)1.7 gene sequence. Accordingly, disclosed hereinare methods of characterizing mutant nucleic acid sequences that encodea Na_(v)1.7 sodium channel alpha-subunit and the use of such nucleicacids to diagnose and treat disease states and neurologic disorders,such as seizures.

In one aspect, disclosed herein is a method of characterizing a nucleicacid sequence that encodes a Na_(v)1.7 sodium channel alpha-subunit,comprising the step of identifying mutations at one or more sites inregions of the nucleic acid sequence that encode various regions of theNa_(v)1.7 sodium channel alpha-subunit. While mutations can be presentin any region of the Na_(v)1.7 nucleic acid sequence, specific regionsof the nucleic acid sequence where mutations can be identified include,but are not limited to, those regions that encode an intracellularN-terminal region, an extracellular loop in domain I, an intracellularloop between domains I and II, an intracellular loop between domains IIand III, an intramembrane region of domain II, or any combinationthereof Such identified nucleotides can indicate the character of thenucleic acid sequence.

The terms “mutation” and “mutant,” as used herein, mean that, at one ormore specific positions in a nucleic acid or amino acid sequence, anucleotide or amino acid that differs from the most commonly foundnucleotide or amino acid can be identified. A mutation includesdeletions, additions, insertions, and substitutions in the nucleotide oramino acid sequence. For example, in one particular mutant Na_(v)1.7nucleic acid sequence disclosed herein, position 184 of the nucleic acidsequence contains a substitution; that is, the most commonly foundnucleotide at position 184 of the Na_(v)1.7 gene is A, whereas in themutant Na_(v)1.7 nucleic acid sequence, the nucleotide found at position184, i.e., the mutated site, is G. One of skill in the art can analyzeposition 184 and determine which of the two amino acids (A or G) ispresent. As another example, in one particular mutant Na_(v)1.7 sodiumchannel alpha-subunit disclosed herein, position 62 of the amino acidsequence contains a substitution; that is, the most commonly found aminoacid at position 62 of the Na_(v)1.7 amino acid sequence is isoleucine,whereas in the mutant Na_(v)1.7 amino acid sequence, the amino acidfound at position 62, i.e., the mutated site, is valine. Also, one ofskill in the art can analyze position 62 of the amino acid sequence anddetermine which of the two amino acids (isoleucine or valine) ispresent. Further, as used herein, “mutant” also includes combinations ofmutations at more than one position in the Na_(v)1.7 nucleic acid oramino acid sequence. Mutations may provide functional differences in thegenetic sequence, through changes in the encoded polypeptide, changes inmRNA stability, binding of transcriptional and translation factors tothe DNA or RNA, and the like. The mutations can also be used as singlenucleotide or single amino acid mutations to detect genetic linkage tophenotypic variation in activity and expression of sodium channels.

As utilized herein, the “character” of the Na_(v)1.7 nucleic acidsequence can be the combination of nucleotides present at mutated sitesthat make up the Na_(v)1.7 sodium channel alpha-subunit haplotype aswell as the biological activity associated with a particular mutation orcombination of mutations.

In one specific aspect, a mutation can be present in the nucleic acidregion encoding the intracellular N-terminus region of the Na_(v)1.7sodium channel alpha-subunit. For example, such a mutation can be at thesite that encodes amino acid residue 62. The mutated site can be atposition 184 of the Na_(v)1.7 nucleic acid sequence. In one particularaspect, the mutation can encode a valine at amino acid residue 62.

In another aspect, a mutation can be present in the nucleic acid regionencoding the extracellular loop of domain I of the Na_(v)1.7 sodiumchannel alpha-subunit. For example, such a mutation can be at the sitethat encodes amino acid residue 149. The mutated site can be at position446 of the Na_(v)1.7 nucleic acid sequence. In one specific aspect, themutation can encode a glutamine at amino acid residue 149.

In yet another aspect, mutations can be present in the nucleic acidregion encoding the intracellular loop between domains I and II of theNa_(v)1.7 sodium channel alpha-subunit. For example, such mutations canbe at sites that encode amino acid residue 641 and/or amino acid residue655. The mutated sites can be at positions 1921 and/or 1964 of theNa_(v)1.7 nucleic acid sequence. In one specific aspect, the mutationcan encode a tyrosine at amino acid residue 641. In another aspect, themutation can encode an arginine at amino acid residue 655.

In a further aspect, a mutation can be present in the nucleic acidregion encoding the intramembrane region of domain II of the Na_(v)1.7sodium channel alpha-subunit. For example, such a mutation can be at thesite that encodes amino acid residue 739. The mutated site can be atposition 2215 of the Na_(v)1.7 nucleic acid sequence. In one specificaspect, the mutation can encode a valine at amino acid residue 739.

In still another aspect, a mutation can be present in the nucleic acidregion encoding the intracellular loop between domains II and III of theNa_(v)1.7 sodium channel alpha-subunit. For example, such a mutation canbe at the site that encodes amino acid residue 1123. The mutated sitecan be at position 3369 of the Na_(v)1.7 nucleic acid sequence. In onespecific aspect, the mutation can encode a phenylalanine at amino acidresidue 1123.

Mutations can also be present in more than one region of the nucleicacid sequence, such as in regions that encode an intracellularN-terminal region and an extracellular loop in domain I; anintracellular N-terminal region and an intracellular loop betweendomains I and II; an intracellular N-terminal region and anintracellular loop between domains II and III; an intracellularN-terminal region and an intramembrane region of domain II; anextracellular loop in domain I and an intracellular loop between domainsI and II; an extracellular loop in domain I and an intracellular loopbetween domains II and III; an extracellular loop in domain I and anintramembrane region of domain II; an intracellular loop between domainsI and II and an intracellular loop between domains II and III; anintracellular loop between domains I and II and an intramembrane regionof domain II; and an intracellular loop between domains II and III andan intramembrane region of domain II.

Some of the mutations that can be identified by the methods disclosedherein include, but are not limited to, mutations at positions 184, 446,1921, 1964, 2215, 3369, or any combination thereof, of the Na_(v)1.7nucleic acid sequence. Any individual mutation can be analyzed at any ofthese positions, or combinations of mutant variants at more than oneposition can be identified and analyzed by the methods disclosed herein.

A number of methods are available for analyzing nucleic acids for thepresence of a specific sequence. For all of the methods describedherein, genomic DNA can be extracted from a sample and this sample canbe from any organism and can be, but is not limited to, peripheralblood, bone marrow specimens, primary tumors, embedded tissue sections,frozen tissue sections, cell preparations, cytological preparations,exfoliate samples (e.g., sputum), fine needle aspirations, amnion cells,fresh tissue, dry tissue, and cultured cells or tissue. Such samples canbe obtained directly from a subject, commercially obtained or obtainedvia other means. Thus, the methods described herein can be utilized toanalyze a nucleic acid sample that comprises genomic DNA, amplified DNA(such as a PCR product), cDNA, cRNA, a restriction fragment or any otherdesired nucleic acid sample. When one performs one of the hereindescribed methods on genomic DNA, typically the genomic DNA will betreated in a manner to reduce viscosity of the DNA and allow bettercontact of a primer or probe with the target region of the genomic DNA.Such reduction in viscosity can be achieved by any desired methods,which are known to the skilled artisan, such as DNase treatment orshearing of the genomic DNA, preferably lightly.

If sufficient DNA is available, genomic DNA can be used directly.Alternatively, the region of interest is cloned into a suitable vectorand grown in sufficient quantity for analysis. The nucleic acid may beamplified by conventional techniques, such as the polymerase chainreaction (PCR), to provide sufficient amounts for analysis. A variety ofPCR techniques are familiar to those skilled in the art. For a review ofPCR technology, see the publication entitled “PCR Methods andApplications” (1991, Cold Spring Harbor Laboratory Press), which isincorporated herein by reference in its entirety for amplificationmethods. In each of these PCR procedures, PCR primers on either side ofthe nucleic acid sequences to be amplified are added to a suitablyprepared nucleic acid sample along with dNTPs and a thermostablepolymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase.The nucleic acid in the sample is denatured and the PCR primers arespecifically hybridized to complementary nucleic acid sequences in thesample. The hybridized primers are extended. Thereafter, another cycleof denaturation, hybridization, and extension is initiated. The cyclesare repeated multiple times to produce an amplified fragment containingthe nucleic acid sequence between the primer sites. PCR has further beendescribed in several patents including U.S. Pat. Nos. 4,683,195,4,683,202, and 4,965,188. Each of these publications is incorporatedherein by reference in its entirety for PCR methods. One of skill in theart would know how to design and synthesize primers flanking any of thenucleic acid sequences disclosed herein.

For example, the disclosed method provides primers GTCCCGCCCATTGCCTGACAC(SEQ ID NO: 20) and TTCTGGTCATGATATGGTTATTCAC (SEQ ID NO: 21), which canbe utilized to amplify the region of the Na_(v)1.7 nucleic acid sequencecomprising nucleotide position 184 in order to identify a mutation atthis site. The disclosed method also provides primersTGATAGATGCGTTGATGACATTGG (SEQ ID NO: 22) and TTCATAAATGCAGTAACTTCCTGG(SEQ ID NO: 23), which can be utilized to amplify the region of theNa_(v)1.7 nucleic acid sequence comprising nucleotide position 446 inorder to identify a mutation at this site. Also, the disclosed methodprovides primers TGTTTCTTTTAAGTCAGTACAGAG (SEQ ID NO: 24) andAGAGCCATTCACAAGACCAGAG (SEQ ID NO: 25), which can be utilized to amplifythe region of the Na_(v)1.7 nucleic acid sequence comprising nucleotideposition 1921 in order to identify a mutation at this site.Additionally, the disclosed method provides primersACTCAGAAAGGCAGAGAGGTG (SEQ ID NO: 26) and TTGCCATGTTATCAATGTCTGTG (SEQID NO: 27), which can be utilized to amplify the region of the Na_(v)1.7nucleic acid sequence comprising nucleotide position 1964 in order toidentify a mutation at this site. Further, the disclosed method providesprimers GACTGATTTGTATCTGGTTAGGAG (SEQ ID NO: 28) andGCAATGTAATTAGGAAGGTGTGAG (SEQ ID NO: 29), which can be utilized toamplify the region of the Na_(v)1.7 nucleic acid sequence comprisingnucleotide position 2215 in order to identify a mutation at this site.For example, the disclosed method provides primersTTTGAATGAACTCTAAATGAACTACC (SEQ ID NO: 30) and TAAGTATTAGGCGTTAAGACAAACC(SEQ ID NO: 31), which can be utilized to amplify the region of theNa_(v)1.7 nucleic acid sequence comprising nucleotide position 3369 inorder to identify a mutation at this site. One of skill in the art wouldknow how to design primers accordingly to amplify any region of theNa_(v)1.7 nucleic acid sequence for the purposes of identifying amutation at any nucleotide position throughout the Na_(v)1.7 sodiumchannel alpha-subunit sequence. Amplification may also be used todetermine whether a mutation is present by using a primer that isspecific for the mutation.

Various methods are known in the art that utilize oligonucleotideligation as a means of detecting mutations, for examples see Riley, etal. (1990) Nucleic Acids Res 18:2887-2890; and Delahunty, et al. (1996)Am J Hum Genet 58:1239-1246, which are incorporated herein by referencein their entirety for methods of detecting mutations. Such methodsinclude single base chain extension (SBCE), oligonucleotide ligationassay (OLA) and cleavase reaction/signal release (Invader methods, ThirdWave Technologies).

LCR and Gap LCR are exponential amplification techniques. Both depend onDNA ligase to join adjacent primers annealed to a DNA molecule. InLigase Chain Reaction (LCR), probe pairs are used which include twoprimary (first and second) and two secondary (third and fourth) probes,all of which are employed in molar excess to target. The first probehybridizes to a first segment of the target strand and the second probehybridizes to a second segment of the target strand, the first andsecond segments being contiguous so that the primary probes abut oneanother in 5′-phosphate-3′-hydroxyl relationship, and so that a ligasecan covalently fuse or ligate the two probes into a fused product. Inaddition, a third (secondary) probe can hybridize to a portion of thefirst probe and a fourth (secondary) probe can hybridize to a portion ofthe second probe in a similar abutting fashion. Of course, if the targetis initially double stranded, the secondary probes also will hybridizeto the target complement in the first instance. Once the ligated strandof primary probes is separated from the target strand, it will hybridizewith the third and fourth probes, which can be ligated to form acomplementary, secondary ligated product. It is important to realizethat the ligated products are functionally equivalent to either thetarget or its complement. By repeated cycles of hybridization andligation, amplification of the target sequence is achieved. A method formultiplex LCR has also been described (WO 9320227, which is incorporatedherein by reference in its entirety for the methods taught therein). GapLCR (GLCR) is a version of LCR where the probes are not adjacent but areseparated by 2 to 3 bases.

A method for typing single nucleotide mutations in DNA, labeled GeneticBit Analysis (GBA), has been described in Nikiforov, et al. (1994)Nucleic Acid Res 22:4167-4175. In this method, specific fragments ofgenomic DNA containing the mutated site(s) are first amplified by thepolymerase chain reaction (PCR) using one regular and onephosphorothioate-modified primer. The double-stranded PCR product isrendered single-stranded by treatment with the enzyme T7 gene 6exonuclease, and captured onto individual wells of a 96 well polystyreneplate by hybridization to an immobilized oligonucleotide primer. Thisprimer is designed to hybridize to the single-stranded target DNAimmediately adjacent from the mutated site of interest. Using the Klenowfragment of E. coli DNA polymerase I or the modified T7 DNA polymerase(Sequenase), the 3′ end of the capture oligonucleotide is extended byone base using a mixture of one biotin-labeled, one fluorescein-labeled,and two unlabeled dideoxynucleoside triphosphates. Antibody conjugatesof alkaline phosphatase and horseradish peroxidase are then used todetermine the nature of the extended base in an ELISA format.Additionally, minisequencing with immobilized primers has been utilizedfor detection of mutations in PCR products (see Pastinen, et al. (1997)Genome Res 7:606-614).

The effect of phosphorothioate bonds on the hydrolytic activity of the5′→3′ double-strand-specific T7 gene 6 exonuclease is used in order toimprove upon GBA. The use of phosphorothioate primers and exonucleasehydrolysis for the preparation of single-stranded PCR products and theirdetection by solid-phase hybridization can be used. (See Nikiforov, etal. (1994) PCR Methods and Applications 3:285-291.) Double-stranded DNAsubstrates containing one phosphorothioate residue at the 5′ end werefound to be hydrolyzed by this enzyme as efficiently as unmodified ones.The enzyme activity was, however, completely inhibited by the presenceof four phosphorothioates. On the basis of these results, a method forthe conversion of double-stranded PCR products into full-length,single-stranded DNA fragments was developed. In this method, one of thePCR primers contains four phosphorothioates at its 5′ end, and theopposite strand primer is unmodified. Following the amplification, thedouble-stranded product is treated with T7 gene 6 exonuclease. Thephosphorothioated strand is protected from the action of this enzyme,whereas the opposite strand is hydrolyzed. When the phosphorothioatedPCR primer is 5′ biotinylated, the single-stranded PCR product can beeasily detected colorimetrically after hybridization to anoligonucleotide probe immobilized on a microtiter plate. A simple andefficient method for the immobilization of relatively shortoligonucleotides to microtiter plates with a hydrophilic surface in thepresence of salt can be used.

DNA analysis based on template hybridization (or hybridization plusenzymatic processing) to an array of surface-bound oligonucleotides iswell suited for high density, parallel, low cost and automatableprocessing (Ives, et al. (1996) Proc SPIE-Int Soc Opt Eng 2680(Ultrasensitive Biochemical Diagnostics) 258-269). Direct fluorescencedetection of labeled DNA provides the benefits of linearity, largedynamic range, multianalyte detection, processing simplicity and safehandling at reasonable cost. The Molecular Tool Corporation has applieda proprietary enzymatic method of solid phase genotyping to DNAprocessing in 96-well plates and glass microscope slides. Detecting thefluor-labeled GBA dideoxynucleotides requires a detection limit ofapproximately 100 mols/μm². Commercially available plate readers detectabout 1000 mols/μm², and an experimental setup with an argon laser andthermoelectrically-cooled CCD can detect approximately 1 order ofmagnitude less signal. The current limit is due to glass fluorescence.Dideoxynucleotides labeled with fluorescein, eosin,tetramethylrhodamine, Lissamine and Texas Red have been characterized,and photobleaching, quenching and indirect detection with fluorogenicsubstrates have been investigated.

Other amplification techniques that can be used in the context of thepresent invention include, but are not limited to, Q-beta amplificationas described in European Patent Application No 4544610, stranddisplacement amplification as described in EP 684 315A and, targetmediated amplification as described in PCT Publication WO 9322461, thedisclosures of which are incorporated herein by reference in theirentirety for the methods taught therein.

Allele specific amplification can also be utilized for biallelicmarkers. Discrimination between the two alleles of a biallelic markercan also be achieved by allele specific amplification, a selectivestrategy, whereby one of the alleles is amplified without amplificationof the other allele. For allele specific amplification, at least onemember of the pair of primers is sufficiently complementary with aregion of a reference sequence (i.e., Na_(v)1.7) comprising thepolymorphic base of a biallelic marker of the present invention tohybridize therewith. Such primers are able to discriminate between thetwo alleles of a biallelic marker. This can be accomplished by placingthe mutated base at the 3′ end of one of the amplification primers. Suchallele specific primers tend to selectively prime an amplification orsequencing reaction so long as they are used with a nucleic acid samplethat contains one of the two alleles present at a biallelic markerbecause the extension forms from the 3′ end of the primer, a mismatch ator near this position has an inhibitory effect on amplification.Therefore, under appropriate amplification conditions, these primersonly direct amplification on their complementary allele. Determining theprecise location of the mismatch and the corresponding assay conditionsare well with the ordinary skill in the art.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g., fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g., 32 P, 35 S, 3 H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g., avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

The sample nucleic acid, e.g., amplified or cloned fragment, can beanalyzed by one of a number of methods known in the art. The nucleicacid can be sequenced by dideoxy or other methods. Hybridization withthe variant sequence can also be used to determine its presence, bySouthern blots, dot blots, etc. The hybridization pattern of a control(reference) and variant sequence to an array of oligonucleotide probesimmobilized on a solid support, as described in U.S. Pat. No. 5,445,934and WO 95/35505, which are incorporated herein by reference in theirentirety for the methods, may also be used as a means of detecting thepresence of variant sequences. Single strand conformational polymorphism(SSCP) analysis, denaturing gradient gel electrophoresis (DGGE),mismatch cleavage detection, and heteroduplex analysis in gel matricesare used to detect conformational changes created by DNA sequencevariation as alterations in electrophoretic mobility. Alternatively,where a mutation creates or destroys a recognition site for arestriction endonuclease (restriction fragment length polymorphism,RFLP), the sample is digested with that endonuclease, and the productssize fractionated to determine whether the fragment was digested.Fractionation is performed by gel or capillary electrophoresis,particularly acrylamide or agarose gels.

The disclosed materials, compositions, and methods also provide the useof the nucleic acid sequences described herein in methods using a mobilesolid support to analyze mutations. See, for example, WO 01/48244, whichis incorporated herein by reference in its entirety for the methodstaught therein.

The method of performing a Luminex FlowMetrix-based SNP analysisinvolves differential hybridization of a PCR product to twodifferently-colored FACS-analyzable beads. The FlowMetrix systemcurrently consists of uniformly-sized 5 micronpolystyrene-divinylbenzene beads stained in eight concentrations of twodyes (orange and red). The matrix of the two dyes in eightconcentrations allows for 64 differently-colored beads that can each bedifferentiated by a FACScalibur suitably modified with the Luminex PCcomputer board. In the Luminex SNP analysis, covalently-linked to a beadis a short (approximately 18-20 bases) “target” oligodeoxynucleotide(oligo). The nucleotide positioned at the center of the target oligoencodes the polymorphic base. A pair of beads are synthesized; each beadof the pair has attached to it one of the polymorphic oligonucleotides.A PCR of the region of DNA surrounding the to-be analyzed SNP isperformed to generate a PCR product. Conditions are established thatallow hybridization of the PCR product preferentially to the bead onwhich is encoded the precise complement. In one format (“withoutcompetitor”), the PCR product itself incorporates a flourescein dye andit is the gain of the flourescein stain on the bead, as measured duringthe FACScalibur run, that indicates hybridization. In a second format(“with competitor,”) the beads are hybridized with a competitor to thePCR product. The competitor itself in this case is labeled withflourescein. And it is the loss of the flourescein by displacement byunlabeled PCR product that indicates successful hybridization.

Isolated Na_(v)1.7 Nucleic Acid Sequences

The nucleic acid sequences disclosed herein can be isolated by methodsknown in the art and described herein. In one aspect, disclosed hereinare isolated nucleic acids comprising nucleotide sequences encodingmutant Na_(v)1.7 sodium channel alpha-subunits. For example, in oneaspect, disclosed herein is an isolated nucleic acid sequence comprisinga nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.In another aspect, disclosed herein is an isolated nucleic acid sequencecomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 3. In yet another aspect, disclosed herein is an isolated nucleicacid sequence comprising a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 4. In a further aspect, disclosed herein is anisolated nucleic acid sequence comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO: 5. In a still further aspect,disclosed herein is an isolated nucleic acid sequence comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6. Inone aspect, disclosed herein is an isolated nucleic acid sequencecomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 7.

Also, disclosed herein is an isolated nucleic acid comprising anucleotide sequence encoding at least 5 contiguous residues of theNa_(v)1.7 sodium channel alpha-subunit. For example, in one aspect,disclosed herein is an isolated nucleic acid comprising a nucleotidesequence encoding at least 5 contiguous residues of the amino acidsequence of SEQ ID NO: 2, wherein one of the amino acid residuescomprises a valine in a position that corresponds to position 62 in SEQID NO: 2. In another aspect, disclosed herein is an isolated nucleicacid comprising a nucleotide sequence encoding at least 5 contiguousresidues of the amino acid sequence of SEQ ID NO: 3, wherein one of theamino acid residues comprises a glutamine in a position that correspondsto position 149 in SEQ ID NO: 3. In yet another aspect, disclosed hereinis an isolated nucleic acid comprising a nucleotide sequence encoding atleast 5 contiguous residues of the amino acid sequence of SEQ ID NO: 4,wherein one of the amino acid residues comprises a tyrosine in aposition that corresponds to position 641 in SEQ ID NO: 4. In a furtheraspect, disclosed herein is an isolated nucleic acid comprising anucleotide sequence encoding at least 5 contiguous residues of the aminoacid sequence of SEQ ID NO: 5, wherein one of the amino acid residuescomprises a arginine in a position that corresponds to position 655 inSEQ ID NO: 5. In a still further aspect, disclosed herein is an isolatednucleic acid comprising a nucleotide sequence encoding at least 5contiguous residues of the amino acid sequence of SEQ ID NO: 6, whereinone of the amino acid residues comprises a valine in a position thatcorresponds to position 739 in SEQ ID NO: 6. In one aspect, disclosedherein is an isolated nucleic acid comprising a nucleotide sequenceencoding at least 5 contiguous residues of the amino acid sequence ofSEQ ID NO: 7, wherein one of the amino acid residues comprises aphenylalanine in a position that corresponds to position 1123 in SEQ IDNO: 7.

Reference Nucleic Acid Sequences

Reference sequences of the Na_(v)1.7 gene comprising the most commonlyfound allele are provided herein. As utilized herein, “referencesequence” refers to a nucleic acid sequence that encodes a Na_(v)1.7sodium channel alpha-subunit or fragment thereof comprising a specificnucleotide at a particular position(s) in the Na_(v)1.7 nucleic acidsequence. Optionally, the reference sequence comprises the most commonlyfound nucleotide or allele at the particular position or positions. Thisreference sequence can be a full-length Na_(v)1.7 nucleic acid sequenceor fragments thereof. An example of a full-length human Na_(v)1.7nucleic acid sequence is provided herein as SEQ ID NO: 1.

The term “wild-type” may also be used to refer to the reference sequencecomprising the most commonly found allele. It will be understood by oneof skill in the art that the designation as “wild-type” is merely aconvenient label for a common allele and should not be construed asconferring any particular property on that form of the sequence.

Alternatively, one of skill in the art can utilize a reference sequenceor a fragment thereof comprising a nucleotide or allele that is not themost commonly found nucleotide or allele at a specific nucleotideposition(s) in the Na_(v)1.7 nucleic acid sequence or can utilize areference sequence that comprises alternative nucleotides at a specificposition(s). An example of a full-length Na_(v)1.7 nucleic acid sequencethat comprises such an alternative nucleotide at position 184 isprovided herein as SEQ ID NO: 8. Therefore, when utilizing thisreference sequence or a fragment thereof, the nucleotide at position 184can be A or G. Other examples of full-length Na_(v)1.7 referencesequences that comprise such alternative nucleotides at positions 446,1921, 1964, 2215, and 3369 are provided herein as SEQ ID NO's: 9, 10,11, 12, and 13, respectively. Therefore, when utilizing these referencesequences or fragments thereof, respectively, the nucleotide at position446 can be C or A, the nucleotide at position 1921 can be A or T, thenucleotide at position 1964 can be position A or G, the nucleotide atposition 2215 can be A or T, and the nucleotide at position 3369 can beG or T.

In one aspect, the reference sequence can comprise a fragment of theNa_(v)1.7 nucleic acid sequence. For example, disclosed herein is areference sequence comprising the nucleotide sequence GCCCTTCATCTATGG(SEQ ID NO: 14), corresponding to nucleotides 177 to 191 of theNa_(v)1.7 gene sequence. This reference sequence has an “A” at position184, which is the most commonly found nucleotide at this position.Therefore, one of skill in the art can compare this reference sequenceto a test sequence and determine if the most commonly found nucleotide(A) is present at position 184 of the test sequence or if anothernucleotide (G) is present at position 184 of the test sequence. Alsoprovided are nucleotide sequence corresponding to any fragment of SEQ IDNO: 14 that includes the A at position 184 or the corresponding sequencewith a G at position 184.

As another example, disclosed herein is a reference sequence comprisingthe nucleotide sequence AACCCGCCGGACTGG (SEQ ID NO: 15), correspondingto nucleotides 439 to 453 of the Na_(v)1.7 gene sequence. This referencesequence has a “C” at position 446, which is the most commonly foundnucleotide at this position. Therefore, one of skill in the art cancompare this reference sequence to a test sequence and determine if themost commonly found nucleotide (C) is present at position 446 of thetest sequence or if another nucleotide (A) is present at position 446 ofthe test sequence. Also provided are nucleotide sequence correspondingto any fragment of SEQ ID NO: 15 that includes the C at position 446 orthe corresponding sequence with a A at position 446.

Also, disclosed herein is a reference sequence comprising the nucleotidesequence GCTCCCCAATGGACA (SEQ ID NO: 16), corresponding to nucleotides1914 to 1928 of the Na_(v)1.7 gene sequence. This reference sequence hasan “A” at position 1921, which is the most commonly found nucleotide atthis position. Therefore, one of skill in the art can compare thisreference sequence to a test sequence and determine if the most commonlyfound nucleotide (A) is present at position 1921 of the test sequence orif another nucleotide (G) is present at position 1921 of the testsequence. Also provided are nucleotide sequence corresponding to anyfragment of SEQ ID NO: 16 that includes the A at position 1921 or thecorresponding sequence with a G at position 1921.

Further, disclosed herein is a reference sequence comprising thenucleotide sequence ATACACAAGAAAAGG (SEQ ID NO: 17), corresponding tonucleotides 1956 to 1971 of the Na_(v)1.7 gene sequence. This referencesequence has an “A” at position 1964, which is the most commonly foundnucleotide at this position. Therefore, one of skill in the art cancompare this reference sequence to a test sequence and determine if themost commonly found nucleotide (A) is present at position 1964 of thetest sequence or if another nucleotide (G) is present at position 1964of the test sequence. Also provided are nucleotide sequencecorresponding to any fragment of SEQ ID NO: 17 that includes the A atposition 1964 or the corresponding sequence with a G at position 1964.

In yet another example, disclosed herein is a reference sequencecomprising the nucleotide sequence TCTTGCAATTACCAT (SEQ ID NO: 18),corresponding to nucleotides 2208 to 2222 of the Na_(v)1.7 genesequence. This reference sequence has an “A” at position 2215, which isthe most commonly found nucleotide at this position. Therefore, one ofskill in the art can compare this reference sequence to a test sequenceand determine if the most commonly found nucleotide (A) is present atposition 2215 of the test sequence or if another nucleotide (G) ispresent at position 2215 of the test sequence. Also provided arenucleotide sequence corresponding to any fragment of SEQ ID NO: 18 thatincludes the A at position 2215 or the corresponding sequence with a Gat position 2215.

In still another example, disclosed herein is a reference sequencecomprising the nucleotide sequence ACCCTTTGCCTGGAG (SEQ ID NO: 19),corresponding to nucleotides 3362 to 3376 of the Na_(v)1.7 genesequence. This reference sequence has a “G” at position 3369, which isthe most commonly found nucleotide at this position. Therefore, one ofskill in the art can compare this reference sequence to a test sequenceand determine if the most commonly found nucleotide (G) is present atposition 3369 of the test sequence or if another nucleotide (T) ispresent at position 3369 of the test sequence. Also provided arenucleotide sequence corresponding to any fragment of SEQ ID NO: 19 thatincludes the G at position 3369 or the corresponding sequence with a Tat position 3369.

Probes and Primers

Nucleic acids of interest comprising the mutations provided herein canbe utilized as probes or primers. The complementary sequences of theNa_(v)1.7 nucleic acid sequences disclosed herein are also provided. Forthe most part, the nucleic acid fragments will be of at least about 15nucleotides, usually at least about 20 nucleotides, often at least about50 nucleotides. Such fragments are useful as primers for PCR,hybridization screening, etc. Larger nucleic acid fragments, forexample, greater than about 100 nucleotides are useful for production ofpromoter fragments, motifs, etc. For use in amplification reactions,such as PCR, a pair of primers will be used. The exact composition ofprimer sequences is not critical to the invention, but for mostapplications the primers will hybridize to the subject sequence understringent conditions, as known in the art.

“Probes,” as used herein, are molecules capable of interacting with atarget nucleic acid, typically in a sequence specific manner, forexample, through hybridization. The hybridization of nucleic acids iswell understood in the art and is discussed herein. Typically, a probecan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art.

By “hybridizing under stringent conditions” or “hybridizing under highlystringent conditions” is meant that the hybridizing portion of thehybridizing nucleic acid, typically comprising at least 15 (e.g., 20,25, 30, or 50 nucleotides), hybridizes to all or a portion of theprovided nucleotide sequence under stringent conditions. The term“hybridization” typically means a sequence driven interaction between atleast two nucleic acid molecules, such as a primer or a probe and agene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.Generally, the hybridizing portion of the hybridizing nucleic acid is atleast 80%, for example, at least 90%, 95%, or 98%, identical to thesequence of or a portion of the Na_(v)1.7 nucleic acid of the invention,or its complement. Hybridizing nucleic acids of the invention can beused, for example, as a cloning probe, a primer (e.g., for PCR), adiagnostic probe, or an antisense probe. Hybridization of theoligonucleotide probe to a nucleic acid sample typically is performedunder stringent conditions. Nucleic acid duplex or hybrid stability isexpressed as the melting temperature or T_(m), which is the temperatureat which a probe dissociates from a target DNA. This melting temperatureis used to define the required stringency conditions. If sequences areto be identified that are related and substantially identical to theprobe, rather than identical, then it is useful to first establish thelowest temperature at which only homologous hybridization occurs with aparticular concentration of salt (e.g., SSC or SSPE). Assuming that a 1%mismatch results in a 1° C. decrease in the T_(m), the temperature ofthe final wash in the hybridization reaction is reduced accordingly (forexample, if sequence having >95% identity with the probe are sought, thefinal wash temperature is decreased by 5° C.). In practice, the changein T_(m) can be between 0.5° C. and 1.5° C. per 1% mismatch. Stringentconditions involve hybridizing at 68° C. in 5×SSC/5× Denhardt'ssolution/1.0% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature.Moderately stringent conditions include washing in 3×SSC at 42° C. Theparameters of salt concentration and temperature can be varied toachieve the optimal level of identity between the probe and the targetnucleic acid. Additional guidance regarding such conditions is readilyavailable in the art, for example, in Sambrook, et al., MolecularCloning, A Laboratory Manual, Cold Spring Harbor Press, New York, N.Y.(1989) ; and Ausubel, et al., eds., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., at Unit 2.10 (1995).

Synthetic analogs of nucleic acids may be preferred for use as probesbecause of superior stability under assay conditions. Modifications inthe native structure, including alterations in the backbone, sugars orheterocyclic bases, have been shown to increase intracellular stabilityand binding affinity. Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage.

Sugar modifications are also used to enhance stability and affinity. Thealpha-anomer of deoxyribose may be used, where the base is inverted withrespect to the natural beta-anomer. The 2′-OH of the ribose sugar may bealtered to form 2′-O-methyl or 2′-O-allyl sugars, which providesresistance to degradation without compromising affinity.

Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5- propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

In one aspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acidcomprising a nucleotide sequence encoding an amino acid sequence of amutated Na_(v)1.7 sodium channel alpha-subunit but not to a nucleic acidsequence that encodes the amino acid sequence of the wild-type Na_(v)1.7sodium channel alpha-subunit. For example, in one aspect, disclosedherein are isolated nucleic acids comprising a sequence that hybridizesunder stringent conditions to a nucleic acid comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 2 but not to thenucleic acid sequence that encodes SEQ ID NO: 38. In another aspect,disclosed herein are isolated nucleic acids comprising a sequence thathybridizes under stringent conditions to a nucleic acid comprising anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 butnot to the nucleic acid sequence that encodes SEQ ID NO: 38. In yetanother aspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acidcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 4 but not to the nucleic acid sequence that encodes SEQ ID NO:38. In an further aspect, disclosed herein are isolated nucleic acidscomprising a sequence that hybridizes under stringent conditions to anucleic acid comprising a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 5 but not to the nucleic acid sequence thatencodes SEQ ID NO: 38. In a still further aspect, disclosed herein areisolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 6 but not to the nucleicacid sequence that encodes SEQ ID NO: 38. In one aspect, disclosedherein are isolated nucleic acids comprising a sequence that hybridizesunder stringent conditions to a nucleic acid comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 7 but not to thenucleic acid sequence that encodes SEQ ID NO: 38.

In another aspect, disclosed herein are isolated nucleic acidscomprising a sequence that hybridizes under stringent conditions to amutated Na_(v)1.7 nucleic acid sequence or a fragment thereof but not toa wild-type Na_(v)1.7 nucleic acid sequence. For example, in one aspect,disclosed herein are isolated nucleic acids comprising a sequence thathybridizes under stringent conditions to a nucleic acid sequence of SEQID NO: 8, or a fragment thereof, such as SEQ ID NO: 14, but not to thenucleic acid sequence of SEQ ID NO: 1. In another aspect, disclosedherein are isolated nucleic acids comprising a sequence that hybridizesunder stringent conditions to a nucleic acid of SEQ ID NO: 9, or afragment thereof, such as SEQ ID NO: 15, but not to the nucleic acidsequence of SEQ ID NO: 1. In yet another aspect, disclosed herein areisolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid sequence of SEQ ID NO: 10, or afragment thereof, such as SEQ ID NO: 16, but not to the nucleic acidsequence of SEQ ID NO: 1. In an further aspect, disclosed herein areisolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid sequence of SEQ ID NO: 11, or afragment thereof, such as SEQ ID NO: 17, but not to the nucleic acidsequence of SEQ ID NO: 1. In a still further aspect, disclosed hereinare isolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid sequence of SEQ ID NO: 12, or afragment thereof, such as SEQ ID NO: 18, but not to the nucleic acidsequence of SEQ ID NO: 1. In one aspect, disclosed herein are isolatednucleic acids comprising a sequence that hybridizes under stringentconditions to a nucleic acid sequence of SEQ ID NO: 13, or a fragmentthereof, such as SEQ ID NO: 19, but not to the nucleic acid sequence ofSEQ ID NO: 1.

In yet another aspect, disclosed herein are isolated nucleic acidsencoding mutant Na_(v)1.7 sodium channels comprising a sequence thathybridizes under stringent conditions to a mutated Na_(v)1.7 nucleicacid comprising a nucleotide sequence encoding an amino acid sequence ofsodium channel alpha-subunit but not to a wild-type Na_(v)1.7 nucleicacid sequence. For example, in one aspect, disclosed herein are isolatednucleic acids comprising a sequence that hybridizes under stringentconditions to a nucleic acid comprising a nucleotide sequence encodingthe amino acid sequence of SEQ ID NO: 2 but not to the nucleic acidsequence of SEQ ID NO: 1. In another aspect, disclosed herein areisolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 3 but not to the nucleicacid sequence of SEQ ID NO: 1. In yet another aspect, disclosed hereinare isolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 4 but not to the nucleicacid sequence of SEQ ID NO: 1. In an further aspect, disclosed hereinare isolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 5 but not to the nucleicacid sequence of SEQ ID NO: 1. In a still further aspect, disclosedherein are isolated nucleic acids comprising a sequence that hybridizesunder stringent conditions to a nucleic acid comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 6 but not to thenucleic acid sequence of SEQ ID NO: 1. In one aspect, disclosed hereinare isolated nucleic acids comprising a sequence that hybridizes understringent conditions to a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 7 but not to the nucleicacid sequence of SEQ ID NO: 1.

In a further aspect, disclosed herein are isolated nucleic acidscomprising a sequence that hybridizes under stringent conditions to amutated Na_(v)1.7 nucleic acid sequence or a fragment thereof but not tothe nucleic acid sequence that encodes SEQ ID NO: 38. For example, inone aspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acidsequence of SEQ ID NO: 8, or a fragment thereof, such as SEQ ID NO: 14,but not to the nucleic acid sequence that encodes SEQ ID NO: 38. Inanother aspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acid ofSEQ ID NO: 9, or a fragment thereof, such as SEQ ID NO: 15, but not tothe nucleic acid sequence that encodes SEQ ID NO: 38. In yet anotheraspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acidsequence of SEQ ID NO: 10, or a fragment thereof, such as SEQ ID NO: 16,but not to the nucleic acid sequence that encodes SEQ ID NO: 38. In anfurther aspect, disclosed herein are isolated nucleic acids comprising asequence that hybridizes under stringent conditions to a nucleic acidsequence of SEQ ID NO: 11, or a fragment thereof, such as SEQ ID NO: 17,but not to the nucleic acid sequence that encodes SEQ ID NO: 38. In astill further aspect, disclosed herein are isolated nucleic acidscomprising a sequence that hybridizes under stringent conditions to anucleic acid sequence of SEQ ID NO: 12, or a fragment thereof, such asSEQ ID NO: 18, but not to the nucleic acid sequence that encodes SEQ IDNO: 38. In one aspect, disclosed herein are isolated nucleic acidscomprising a sequence that hybridizes under stringent conditions to anucleic acid sequence of SEQ ID NO: 13, or a fragment thereof, such asSEQ ID NO: 19, but not to the nucleic acid sequence that encodes SEQ IDNO: 38.

Arrays

The disclosed materials, compounds, and methods also provide an array ofoligonucleotides for identification of mutations, where discretepositions on the array are complementary to one or more of the providedmutated sequences, e.g. oligonucleotides of at least 12 nucleotides,frequently 15 nucleotides, 20 nucleotides, or larger, and including thesequence flanking the mutated position. Such an array may comprise aseries of oligonucleotides, each of which can specifically hybridize toa different mutation of the disclosed compositions. An array maycomprise all or a subset of nucleic acid sequences having SEQ ID NOs: 8,9, 10, 11, 12, and/or 13, or any fragment of at least 15 contiguousnucleotides thereof, for example SEQ ID NOs: 14, 15, 16, 17, 18, and/or19. Usually such an array will include at least 2 different mutatedsequences, i.e., mutations located at unique positions within the locus,and may include all of the provided mutations. Therefore, the array caninclude wild-type sequences comprising the most commonly found alleles.The array can optionally comprise the most commonly found allele at afirst, second, third, fourth, fifth, or more positions as well as othernucleotides at each of these positions. Each oligonucleotide sequence onthe array will usually be at least about 12 nucleotides in length (i.e.,10-15 nucleotides), may be the length of the provided mutated sequences,or may extend into the flanking regions to generate fragments of 100 to200 nucleotides in length. For examples of arrays, see Ramsay, (1998)Nat Biotech 16:4044; Hacia, et al. (1996) Nature Genetics 14:441-447;Lockhart, et al. (1996) Nature Biotechnol 14:1675-1680; and De Risi, etal. (1996) Nature Genetics 14:457-460, which are incorporated byreference in their entirety for the methods of making and using arrays.

Haplotyping

In another aspect, the disclosed materials, compositions, articles,devices, and methods relate to a method for determining a Na_(v)1.7haplotype in a human subject, wherein the method comprises identifyingone or more nucleotides encoding amino acid residues 62, 149, 641, 655,739, 1123, or any combination thereof, wherein the nucleotide ornucleotides indicate the haplotype. The disclosed subject matter alsoprovides a method for determining a Na_(v)1.7 haplotype in a humansubject comprising identifying one or more nucleotides present at one ormore of sites 184, 446, 1921, 1964, 2215, or 3369, in either or bothcopies of the Na_(v)1.7 gene contained in the subject genomic nucleicacid, wherein the nucleotide present at the mutated site or sitesindicates the Na_(v)1.7 haplotype. It will be recognized by one of skillin the art that numerous haplotypes are possible.

For example, one of skill in the art could identify the nucleotidepresent in either or both copies of the Na_(v)1.7 gene contained in thesubject genomic nucleic acid that encodes for amino acid 62 in theNa_(v)1.7 sodium channel alpha-subunit sequence. The haplotypes for thisparticular analysis can be I62V, P149Q, N641Y, K655R, I739V, L1123F, orany combination thereof, where the number indicates a position in theNa_(v)1.7 sodium channel alpha-subunit, the first letter represents themost common amino acid found at that positions, and the last letterrepresents the amino acid found in the haplotype. Similarly, one ofskill in the art could identify the nucleotide in a Na_(v)1.7 nucleicacid sequence at position 184, 446, 1921, 1964, 2215, and/or 3369, anddetermine the Na_(v)1.7 haplotype. Therefore, any of positions 184, 446,1921, 1964, 2215, and/or 3369 in the nucleic acid sequence or positions62, 149, 641, 655, 739, and/or 1123 in the encoded amino acid sequencecan be analyzed individually or in combination to obtain the haplotypesof the disclosed subject matter.

Determining a Predisposition

Disclosed herein is a method for determining a subject's predispositionto a neurologic disorder associated with a sodium channel mutationcomprising comparing the subject's Na_(v)1.7 haplotype with one or morereference haplotypes that correlate with the neurologic disorder, asimilar haplotype in the subject's Na_(v)1.7 haplotype as compared tothe reference haplotype or haplotypes indicating a predisposition to theneurologic disorder.

As used herein, “neurologic disorder associated with a sodium channelmutation” includes, but is not limited to, seizure disorders (e.g.,febrile seizures, nonfebrile seizures, and epileptic seizures). As usedherein “epliptic seizures” includes, but is not limited to, partial(e.g., simple and complex) and generalized (e.g., absence, myoclonic,and tonic-clonic) seizures, temporal lobe epilepsy, and severe myoclonicepilepsy of infancy.

Each haplotype can be correlated with specific neurologic disorders orseverity of such disorders to generate a database of referencehaplotypes, such that one of skill in the art can compare a subject'shaplotype to a reference haplotype or haplotypes and determine whetherthe subject is at risk for a neurologic disorder.

The reference haplotype can comprise nucleotides that encode one or moremutations in the Na_(v)1.7 sodium channel alpha-subunit. For example,the reference haplotype can comprise nucleotides that encode one or moremutations at residue 62, residue 149, residue 641, residue 655, residue739, or residue 1123 of the encoded amino acid sequence of Na_(v)1.7.

Since subjects will vary depending on numerous parameters including, butnot limited to, race, age, weight, medical history etc., as moreinformation is gathered on populations, the database can containhaplotype information classified by race, age, weight, medical historyetc., such that one of skill in the art can assess the subject's risk ofdeveloping neurologic disorders based on information more closelyassociated with the subject's demographic profile. Where there is adifferential distribution of a mutation by racial background or anotherparameter, guidelines for drug administration can be generally tailoredto a particular group.

It will be appreciated by those skilled in the art that the nucleicacids provided herein as well as the nucleic acid and amino acidsequences identified from subjects can be stored, recorded, andmanipulated on any medium which can be read and accessed by a computer.As used herein, the words “recorded” and “stored” refer to a process forstoring information on a computer medium. A skilled artisan can readilyadopt any of the presently known methods for recording information on acomputer readable medium to generate a list of sequences comprising oneor more of the nucleic acids of the invention. Another aspect of thepresent invention is a computer readable medium having recorded thereonat least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 250, 300, 400, 500,1000, 2000, 3000, 4000 or 5000 nucleic acids of the invention or nucleicacid sequences identified from subjects.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disc, afloppy disc, a magnetic tape, CD-ROM, DVD, RAM, or ROM as well as othertypes of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularlycomputer systems which contain the sequence information describedherein. As used herein, “a computer system” refers to the hardwarecomponents, software components, and data storage components used tostore and/or analyze the nucleotide sequences of the present inventionor other sequences. The computer system preferably includes the computerreadable media described above, and a processor for accessing andmanipulating the sequence data.

Preferably, the computer is a general purpose system that comprises acentral processing unit (CPU), one or more data storage components forstoring data, and one or more data retrieving devices for retrieving thedata stored on the data storage components. A skilled artisan canreadily appreciate that any one of the currently available computersystems are suitable.

In one particular aspect, the computer system includes a processorconnected to a bus which is connected to a main memory, preferablyimplemented as RAM, and one or more data storage devices, such as a harddrive and/or other computer readable media having data recorded thereon.In some embodiments, the computer system further includes one or moredata retrieving devices for reading the data stored on the data storagecomponents. The data retrieving device may represent, for example, afloppy disk drive, a compact disk drive, a magnetic tape drive, a harddisk drive, a CD-ROM drive, a DVD drive, etc. In some embodiments, thedata storage component is a removable computer-readable medium such as afloppy disk, a compact disk, a magnetic tape, etc. containing controllogic and/or data recorded thereon. The computer system mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. Software foraccessing and processing the nucleotide sequences of the nucleic acidsof the invention (such as search tools, compare tools, modeling tools,etc.) may reside in main memory during execution.

In some aspects, the computer system may further comprise a sequencecomparer for comparing the nucleic acid sequences stored on a computerreadable medium to another test sequence stored on a computer readablemedium. A “sequence comparer” refers to one or more programs which areimplemented on the computer system to compare a nucleotide sequence withother nucleotide sequences.

Accordingly, one aspect of the present invention is a computer systemcomprising a processor, a data storage device having stored thereon anucleic acid of the invention, a data storage device having retrievablystored thereon reference nucleotide sequences to be compared with testor sample sequences and a sequence comparer for conducting thecomparison. The sequence comparer may indicate a homology level betweenthe sequences compared or identify a difference between the twosequences. For example, a reference sequence comprising SEQ ID NO: 8 orany fragment thereof, such as SEQ ID NO: 14, can be compared with a testsequence from a subject to determine if the test sequence is the same asthe reference sequence, e.g., contains an A at position 184 or adifferent nucleotide (G).

Alternatively, the computer program may be a computer program whichcompares a test nucleotide sequence(s) from a subject or a plurality ofsubjects to a reference nucleotide sequence(s) in order to determinewhether the test nucleotide sequence(s) differs from or is the same as areference nucleic acid sequence(s) at one or more positions. Optionallysuch a program records the length and identity of inserted, deleted orsubstituted nucleotides with respect to the sequence of either thereference polynucleotide or the test nucleotide sequence. In oneembodiment, the computer program may be a program which determineswhether the nucleotide sequences of the test nucleotide sequencecontains one or more single nucleotide mutations with respect to areference nucleotide sequence. These single nucleotide mutations mayeach comprise a single base substitution, insertion, or deletion.

Accordingly, another aspect of the materials, compounds, articles,devices, and methods disclosed herein is a method for determiningwhether a test nucleotide sequence differs at one or more nucleotidesfrom a reference nucleotide sequence comprising the steps of reading thetest nucleotide sequence and the reference nucleotide sequence throughuse of a computer program which identifies differences between nucleicacid sequences and identifying differences between the test nucleotidesequence and the reference nucleotide sequence with the computerprogram. The computer program can be a program which identifies singlenucleotide polymorphisms. The method may be implemented by the computersystems described above. The method may also be performed by reading atleast 2, 5, 10, 15, 20, 25, 30, 50, 100, or more test nucleotidesequences and the reference nucleotide sequences through the use of thecomputer program and identifying differences between the test nucleotidesequences and the reference nucleotide sequences with the computerprogram. A computer program that identifies single nucleotide mutationsin a Na_(v)1.7 gene sequence and determines a subject's haplotype isalso contemplated by the subject matter disclosed herein. The subjectmatter disclosed herein also provides for a computer program thatcorrelates haplotypes with Na_(v)1.7 levels such that one of skill inthe art can assess a subject's risk of developing a neurologic disorder,such as febrile seizures, nonfebrile seizures, or epileptic seizures.The computer program can optionally include treatment options or drugindications for subjects with haplotypes associated with increased riskof seizures.

The nucleic acids of the invention (both test nucleic acid sequences andreference nucleic acid sequences) may be stored and manipulated in avariety of data processor programs in a variety of formats. For example,they may be stored as text in a word processing file, such asMicrosoftWORD or WORDPERFECT or as an ASCII file in a variety ofdatabase programs familiar to those of skill in the art, such as DB2,SYBASE, or ORACLE. In addition, many computer programs and databases maybe used as sequence comparers, identifiers, or sources of referencenucleotide sequences. The following list is intended not to limit theinvention but to provide guidance to programs and databases which areuseful with the nucleic acid sequences of the invention. The programsand databases which may be used include, but are not limited to:MacPattern (EMBL), DiscoveryBase (Molecular Applications Group),GeneMine (Molecular Applications Group), Look (Molecular ApplicationsGroup), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI),BLASTN and BLASTX (Altschul, et al. (1990) J Mol Biol 3:403-410), FASTA(Pearson and Lipman, (1988) Proc Natl Acad Sci USA 85:2444-2448), FASTDB(Brutlag et al., (1990) Compt Appl Biosci 6:237-245), Catalyst(Molecular Simulations Inc.), Catalyst/SHAPE (Molecular SimulationsInc.), Cerius.sup.2.DBAccess (Molecular Simulations Inc.), HypoGen(Molecular Simulations Inc.), Insight II (Molecular Simulations Inc.),Discover (Molecular Simulations Inc.), CHARMm (Molecular SimulationsInc.), Felix (Molecular Simulations Inc.), DelPhi, (MolecularSimulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology(Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS(Molecular Simulations Inc.), Quanta/Protein Design (MolecularSimulations Inc.), WebLab (Molecular Simulations Inc.), WebLab DiversityExplorer (Molecular Simulations Inc.), Gene Explorer (MolecularSimulations Inc.), SeqFold (Molecular Simulations Inc.), theEMBL/Swissprotein database, the MDL Available Chemicals Directorydatabase, the MDL Drug Data Report data base, the ComprehensiveMedicinal Chemistry database, Derwents's World Drug Index database, theBioByteMasterFile database, the Genbank database, and the Genseqndatabase. Many other programs and data bases would be apparent.

Delivery of the Na_(v)1.7 Nucleic Acid Sequence

Optionally, the nucleic acids described herein are delivered to variousexpression systems. There are a number of compositions and methods whichcan be used to deliver nucleic acids to cells, either in vitro or invivo. These methods and compositions can largely be broken down into twoclasses: viral based delivery systems and non-viral based deliverysystems. For example, the nucleic acids can be delivered through anumber of direct delivery systems such as, electroporation, lipofection,calcium phosphate precipitation, plasmids, viral vectors, viral nucleicacids, phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, et al. (1990)Science 247:1465-1468; and Wolff, (1991) Nature 352:815-818. Suchmethods are well known in the art and readily adaptable for use with thecompositions and methods described herein. In certain cases, the methodswill be modified to specifically function with large DNA molecules.

Nucleic Acid Based Delivery Systems: Vectors

In one aspect, disclosed herein are expression vectors comprising anucleic acid comprising a nucleotide sequence encoding an amino acidsequence of mutated Na_(v)1.7 sodium channel alpha-subunit wherein thenucleotide sequence is operably linked to an expression controlsequence. For example, in one aspect, disclosed herein are expressionvectors comprising a nucleic acid comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO: 2 operably linked to anexpression control sequence. In another aspect, disclosed herein areexpression vectors comprising a nucleic acid comprising a nucleotidesequence encoding the amino acid sequence of SEQ ID NO: 3 operablylinked to an expression control sequence. In yet another aspect,disclosed herein are expression vectors comprising a nucleic acidcomprising a nucleotide sequence encoding the amino acid sequence of SEQID NO: 4 operably linked to an expression control sequence. In a furtheraspect, disclosed herein are expression vectors comprising a nucleicacid comprising a nucleotide sequence encoding the amino acid sequenceof SEQ ID NO: 5 operably linked to an expression control sequence. In astill further aspect, disclosed herein are expression vectors comprisinga nucleic acid comprising a nucleotide sequence encoding the amino acidsequence of SEQ ID NO: 6 operably linked to an expression controlsequence. In one aspect, disclosed herein are expression vectorscomprising a nucleic acid comprising a nucleotide sequence encoding theamino acid sequence of SEQ ID NO: 7 operably linked to an expressioncontrol sequence.

Further provided are expression vectors comprising any fragment of thenucleic acid encoding SEQ ID NOs: 2-7. Such fragments preferably encodeat least 5 contiguous amino acid sequences of SEQ ID NOs: 2-7.

Expression or transfer vectors can be any nucleotide construction usedto deliver genes into cells (e.g., a plasmid), or as part of a generalstrategy to deliver genes, e.g., as part of recombinant retrovirus oradenovirus (Ram, et al. (1993) Cancer Res 53:83-88).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as SEQ ID NOs: 8, 9, 10, 11, 12, and/or 13into the cell without degradation and include a promoter yieldingexpression of the gene in the cells into which it is delivered. Viralvectors are, for example, Adenovirus, Adeno-associated virus, Herpesvirus, Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus,Sindbis and other RNA viruses, including those viruses with the HIVbackbone. Also preferred are any viral families which share theproperties of these viruses which make them suitable for use as vectors.Retroviruses include Murine Maloney Leukemia virus, MMLV, andretroviruses that express the desirable properties of MMLV as a vector.Retroviral vectors are able to carry a larger genetic payload, i.e., atransgene or marker gene, than other viral vectors, and for this reasonare a commonly used vector. However, they are not as useful innon-proliferating cells. Adenovirus vectors are relatively stable andeasy to work with, have high titers, and can be delivered in aerosolformulation, and can transfect non-dividing cells. Pox viral vectors arelarge and have several sites for inserting genes, they are thermostableand can be stored at room temperature. In one specific aspect is a viralvector that has been engineered so as to suppress the immune response ofthe host organism, elicited by the viral antigens. Vectors of this typewill carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase III transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, Retroviralvectors for gene transfer. In Microbiology-1985, American Society forMicrobiology, pp. 229-232, Washington, (1985), which is incorporated byreference herein in its entirety for retroviral vectors and methods ofmaking them. Examples of methods for using retroviral vectors for genetherapy are described in U.S. Pat. Nos. 4,868,116 and 4,980,286; PCTapplications WO 90/02806 and WO 89/07136; and Mulligan, (Science260:926-932 (1993)); the teachings of which are incorporated byreference herein in its entirety for retroviral vectors and methods ofusing them.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner, et al. (1987) J Virology 61:1213-1220; Massie, etal. (1986) Mol Cell Biol 6:2872-2883; Haj-Ahmad, et al. (1986) JVirology 57:267-274; Davidson, et al. (1987) J Virology 61:1226-1239;Zhang, (1993) BioTechniques 15:868-872). The benefit of the use of theseviruses as vectors is that they are limited in the extent to which theycan spread to other cell types, since they can replicate within aninitial infected cell, but are unable to form new infectious viralparticles. Recombinant adenoviruses have been shown to achieve highefficiency gene transfer after direct, in vivo delivery to airwayepithelium, hepatocytes, vascular endothelium, CNS parenchyma and anumber of other tissue sites (Morsy, (1993) J Clin Invest 92:1580-1586;Kirshenbaum, (1993) J Clin Invest 92:381-387; Roessler, (1993) J ClinInvest 92:1085-1092; Moullier, (1993) Nature Genetics 4:154-159; LaSalle, (1993) Science 259:988-990; Gomez-Foix, (1992) J Biol Chem267:25129-25134; Rich, (1993) Human Gene Therapy 4:461-476; Zabner,(1994) Nature Genetics 6:75-83; Guzman, (1993) Circulation Res73:1201-1207; Bout, (1994) Human Gene Therapy 5:3-10; Zabner, (1993)Cell 75:207-216; Caillaud, (1993) Eur. J. Neuroscience 5:1287-1291;Ragot, (1993) J Gen Virology 74:501-507). Recombinant adenovirusesachieve gene transduction by binding to specific cell surface receptors,after which the virus is internalized by receptor-mediated endocytosis,in the same manner as wild type or replication-defective adenovirus(Chardonnet, et al. (1970) Virology 40:462-477; Brown, et al. (1973) JVirology 12:386-396; Svensson, et al. (1985) J Virology 55:442-449;Seth, et al. (1985) J Virol 51:650-655; Seth, et al. (1984) Mol CellBiol 4:1528-1533; Varga, et al. (1991) J Virology 65:6061-6070; Wickham,et al. (1993) Cell 73:309-319).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another aspect, both the E1 and E3 genes areremoved from the adenovirus genome.

Adeno-Associated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereof,confer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is incorporated by reference herein in its entirety formaterial related to the AAV vector.

The disclosed vectors described throughout thus provide nucleic acidswhich are capable of integration into a mammalian chromosome withoutsubstantial toxicity. The vectors can also provide nucleic acids thatcan be expressed in oocytes (including, e.g., Kenopus oocytes).

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of nucleicacids that function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun, et al. (1994) Nature Genetics 8:33-41; Cotter, etal. (1999) Curr Opin Mol Ther 5:633-644). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable. Themaintenance of these episomes requires a specific EBV nuclear protein,EBNA1, constitutively expressed during infection with EBV. Additionally,these vectors can be used for transfection, where large amounts ofprotein can be generated transiently in vitro. Herpesvirus ampliconsystems are also being used to package pieces of DNA>220 kb and toinfect cells that can stably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

Non-Nucleic Acid Based Systems

The disclosed compositions can also be delivered to the target cells ina variety of ways other than through nucleic acid based methods. Forexample, the compositions can be delivered through electroporation, orthrough lipofection, or through calcium phosphate precipitation. Thedelivery mechanism chosen will depend in part on the type of celltargeted and whether the delivery is occurring for example in vivo or invitro.

Thus, the compositions can comprise, in addition to the disclosed mutantNa_(v)1.7 nucleic acid sequences or vectors, for example, lipids such asliposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DCcholesterol) or anionic liposomes. Liposomes can further compriseproteins to facilitate targeting a particular cell, if desired.Administration of a composition comprising a compound and a cationicliposome can be administered to the blood afferent to a target organ orinhaled into the respiratory tract to target cells of the respiratorytract. Regarding liposomes, see, e.g., Brigham, et al. (1989) Am J RespCell Mol Biol 1:95-100; Felgner, et al. (1987) Proc Natl Acad Sci USA84:7413-7417; U.S. Pat. No. 4,897,355. Furthermore, the compound can beadministered as a component of a microcapsule that can be targeted tospecific cell types, such as macrophages, or where the diffusion of thecompound or delivery of the compound from the microcapsule is designedfor a specific rate or dosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al. (1991)Bioconjugate Chem. 2:447-451; Bagshawe, et al. (1998) Br J Cancer60:275-281; Bagshawe, et al. (1988) Br J Cancer 58:700-703; Senter, etal. (1993) Bioconjugate Chem 4:3-9; Battelli, et al. (1992) CancerImmunol Immunother 35:421-425; Pietersz, et al. (1992) Immunolog Rev129:57-80; Roffler, et al. (1991) Biochem Pharmacol 42:2062-2065). Thesetechniques can be used for a variety of other specific cell types.Vehicles such as “stealth” and other antibody conjugated liposomes(including lipid mediated drug targeting to colonic carcinoma), receptormediated targeting of DNA through cell specific ligands, lymphocytedirected tumor targeting, and highly specific therapeutic retroviraltargeting of murine glioma cells in vivo. The following references areexamples of the use of this technology to target specific proteins totumor tissue (Hughes, et al. (1989) Cancer Res 49:6214-6220; Litzinger,et al. (1992) Biochimica et Biophysica Acta 1104:179-187). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin coated pits,enter the cell via clathrin coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor mediatedendocytosis has been reviewed (see Brown, et al. (1991) DNA and CellBiology 10:399-409).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

Expression

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain expression control sequences,i.e., promoters, and/or enhancers to help control the expression of thedesired gene product. A promoter is generally a sequence or sequences ofDNA that function when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g., beta-actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers, et al. (1978) Nature 273: 113). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (see Greenway, et al. (1982) Gene18:355-360). Of course, promoters from the host cell or related speciesalso are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, et al. (1981) Proc Natl Acad Sci USA 78:993) or 3′ (Lusky, etal. (1983) Mol Cell Bio 3:1108) to the transcription unit. Furthermore,enhancers can be within an intron (Banerji, et al. (1983) Cell 33:729)as well as within the coding sequence itself (Osborne, et al. (1984) MolCell Bio 4:1293). They are usually between 10 and 300 bp in length, andthey function in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (e.g.,globin, elastase, albumin, fetoprotein, and insulin), typically, onewill use an enhancer from a eukaryotic cell virus for generalexpression. Preferred examples are the SV40 enhancer on the late side ofthe replication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments, the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full-length promoter), and retroviral vector LTF.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. For example, the glialfibrillary acetic protein (GFAP) promoter has been used to selectivelyexpress genes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′-untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodesbeta-galactosidase, and green fluorescent protein (GFP).

Marker product, as used herein, is synonymous with “reporter protein.”As used herein, a “reporter protein” is any protein that can bespecifically detected when expressed. Reporter proteins are useful fordetecting or quantifying expression from expression sequences. Manyreporter proteins are known to one of skill in the art. These include,but are not limited to, beta-galactosidase, luciferase, and alkalinephosphatase that produce specific detectable products. Fluorescentreporter proteins can also be used, such as green fluorescent protein(GFP), green reef coral fluorescent protein (G-RCFP), cyan fluorescentprotein (CFP), red fluorescent protein (RFP) and yellow fluorescentprotein (YFP).

In some embodiments the marker or reporter protein may be a selectablemarker. Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycinanalog G418, hydromycin, and puromycin. When such selectable markers aresuccessfully transferred into a mammalian host cell, the transformedmammalian host cell can survive if placed under selective pressure.There are two widely used distinct categories of selective regimes. Thefirst category is based on a cell's metabolism and the use of a mutantcell line which lacks the ability to grow independent of a supplementedmedia. Two examples are: CHO DHFR cells and mouse LTK cells. These cellslack the ability to grow without the addition of such nutrients asthymidine or hypoxanthine. Because these cells lack certain genesnecessary for a complete nucleotide synthesis pathway, they cannotsurvive unless the missing nucleotides are provided in a supplementedmedia. An alternative to supplementing the media is to introduce anintact DHFR or TK gene into cells lacking the respective genes, thusaltering their growth requirements. Individual cells which were nottransformed with the DHFR or TK gene will not be capable of survival innon supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern, et al. (1982)J Molec Appl Genet 1:327), mycophenolic acid, (Mulligan, et al. (1980)Science 209:1422) or hygromycin, (Sugden, et al. (1985) Mol Cell Biol5:410-413). The three examples employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.Others include the neomycin analog G418 and puramycin.

Cultured Cells

The materials, compositions, articles, devices, and methods disclosedherein, in one aspect, related to a method of making a mutant Nav1.7sodium channel alpha subunit comprising culturing the cells comprisingvectors comprising mutant Na_(v)1.7 nucleic acids under conditionsallowing expression of the polypeptide encoded by the nucleic acid,wherein the polypeptide comprises a mutant Na_(v)1.7 sodium channel.

Transgenic Animals

In one aspect, disclosed herein are transgenic animals that express oneor more of the mutant Na_(v)1.7 sodium channels described herein. Forexample, disclosed herein is a transgenic mouse comprising cells thatencode a mutant Na_(v)1.7 sodium channel alpha-subunit, wherein themouse exhibits increased seizure activity as compared to the wild-typeanimal.

“Transgenic animal” is used herein to mean an animal comprising atransgene. By a “transgene” is meant a nucleic acid sequence that isinserted by artifice into a cell and becomes a part of the genome ofthat cell and its progeny. Such a transgene may be (but is notnecessarily) partly or entirely heterologous (for example, derived froma different species) to the cell. A transgenic animal can be anynon-human animal, such as a mouse, rat, guinea pig, sheep, pig, goat,and the like. Transgenic animals are made by techniques that are wellknown in the art. For example, a transgenic animal can be prepared bythe method used in U.S. Pat. No. 4,736,866

Mutant Na_(v)1.7 Sodium Channel Alpha-Subunits

In one aspect, disclosed herein are mutant Na_(v)1.7 sodium channelalpha-subunits and the use of such mutant Na_(v)1.7 sodium channels todiagnose and treat disease states such as, for example, neurologicdisorders associated with a sodium channel mutation. It was found thatspecific sites in the Na_(v)1.7 sodium channel alpha-subunit aremutated, i.e., the amino acid at a specific position or at specificpositions differs from that observed in the most commonly foundNa_(v)1.7 sodium channel.

As this specification discusses various amino acid sequences it isunderstood that the nucleic acids that can encode those amino acidsequences are also disclosed. This would include all degeneratesequences related to a specific amino acid sequence, i.e. all nucleicacids having a sequence that encodes one particular amino acid sequenceas well as all nucleic acids, including degenerate nucleic acids,encoding the disclosed variants and derivatives of the amino acidsequences. Thus, while each particular nucleic acid sequence may not bewritten out herein, it is understood that each and every sequence is infact disclosed and described herein through the disclosed amino acidsequence. For example, one of the many nucleic acid sequences that canencode the amino acid sequence of SEQ ID NO: 2 is set forth in SEQ IDNO: 8. Another nucleic acid sequence that encodes the amino acidsequence of SEQ ID NO: 3 is set forth in SEQ ID NO: 9. Another nucleicacid sequence that encodes the amino acid sequence of SEQ ID NO: 4 isset forth in SEQ ID NO: 10. Another nucleic acid sequence that encodesthe amino acid sequence of SEQ ID NO: 5 is set forth in SEQ ID NO: 11.Another nucleic acid sequence that encodes the amino acid sequence ofSEQ ID NO: 6 is set forth in SEQ ID NO: 12. Another nucleic acidsequence that encodes the amino acid sequence of SEQ ID NO: 7 is setforth in SEQ ID NO: 13. It is also understood that while no amino acidsequence indicates what particular DNA sequence encodes that proteinwithin an organism, where particular variants of a disclosed protein aredisclosed herein, the known nucleic acid sequence that encodes thatamino acid sequence in the particular mutant Na_(v)1.7 sodium channelalpha-subunit from which that amino acid sequence arises is also knownand herein disclosed and described.

In one aspect, the mutant Na_(v)1.7 sodium channel alpha-subunitsdescribed herein have one or more mutated sites. For example, in oneaspect, disclosed herein is a mutant Na_(v)1.7 sodium channelalpha-subunit where the amino acid at position 62 is not isoleucine (I)as is commonly found at position 62 but, rather, valine (V) (SEQ ID NO:2). In another aspect, disclosed herein is a mutant Na_(v)1.7 sodiumchannel alpha-subunit where the amino acid at position 149 is notproline (P) as is commonly found at position 149 but, rather, glutamine(Q) (SEQ ID NO: 3). In another aspect, disclosed herein is a mutantNa_(v)1.7 sodium channel alpha-subunit where the amino acid at position641 is not asparagines (N) as is commonly found at position 641 but,rather, tyrosine (Y) (SEQ ID NO: 4). In yet another aspect, disclosedherein is a mutant Na_(v)1.7 sodium channel alpha-subunit where theamino acid at position 655 is not lysine (K) as is commonly found atposition 655 but, rather, arginine (R) (SEQ ID NO: 5). In a furtheraspect, disclosed herein is a mutant Na_(v)1.7 sodium channelalpha-subunit where the amino acid at position 739 is not isoleucine (I)as is commonly found at position 739 but, rather, valine (V) (SEQ ID NO:6). In a still further aspect, disclosed herein is a mutant Na_(v)1.7sodium channel alpha-subunit where the amino acid at position 1123 isnot leucine (L) as is commonly found at position 1123 but, rather,phenylalanine (F) (SEQ ID NO: 7).

Also contemplated are variants and derivatives of the disclosed mutantNa_(v)1.7 amino acid sequences. It is understood that one way to definethe variants and derivatives of the disclosed proteins herein is throughdefining the variants and derivatives in terms of homology/identity tospecific known sequences. For example, SEQ ID NO: 2 sets forth aparticular sequence of a mutant I62V mutant sodium channelalpha-subunit, SEQ ID NO: 3 sets forth a particular sequence of a mutantP149Q Na_(v)1.7 sodium channel alpha-subunit, SEQ ID NO: 4 sets forth aparticular sequence of a mutant N641Y Na_(v)1.7 sodium channelalpha-subunit, SEQ ID NO: 5 sets forth a particular sequence of a mutantK655R Na_(v)1.7 sodium channel alpha-subunit, SEQ ID NO: 6 sets forth aparticular sequence of a mutant I739V Na_(v)1.7 sodium channelalpha-subunit, and SEQ ID NO: 7 sets forth a particular sequence of amutant L1123F Na_(v)1.7 sodium channel alpha-subunit. Specificallydisclosed are variants of these and other proteins herein disclosedwhich have at least, 70% or 75% or 80% or 85% or 90% or 95% homology tothe stated sequence. Also, provided are amino acid sequences comprisingthe sequences of SEQ ID NOs: 2, 3, 4, 5, 6, and 7, or any fragmentthereof wherein the sequence comprises one or more conservative aminoacid substitutions. Preferably, the amino acid sequence withconservative amino acid substitutions maintains sodium channel function.Examples of conservative amino acid substitutions are shown in Table 1.Those of skill in the art readily understand how to determine thehomology of two proteins. For example, the homology can be calculatedafter aligning the two sequences so that the homology is at its highestlevel.

TABLE 1 Amino Acid Substitutions Original Residue Exemplary ConservativeSubstitutions, others are known in the art. Ala

 ser Arg

 lys or gln Asn

 gln or his Asp

 glu Cys

 ser Gln

 asn or lys Glu

 asp Gly

 pro His

 asn or gln Ile

 leu or val Leu

 ile or val Lys

 arg or gln; Met

 leu or ile Phemet

 leu or tyr Ser

 thr Thr

 ser Trp

 tyr Tyr

 trp or phe Val

 ile or leu

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv Appl Math 2:482, by the homology alignment algorithm of Needlemanand Wunsch (1970) J Mol Biol 48:443, by the search for similarity methodof Pearson and Lipman, (1988) Proc Natl Acad Sci USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection.

Also, disclosed herein are isolated polypeptides and fragments ofpolypeptides comprising mutant Na_(v)1.7 sodium channel alpha-subunitamino acid sequences. For example, disclosed herein are isolatedpolypeptides having amino acid sequences of SEQ ID NOs: 2, 3, 4, 5, 6,and 7. In another aspect, disclosed herein are fragments of suchsequences. For example, disclosed herein are isolated polypeptideshaving amino acid sequences of SEQ ID NOs: 32, 33, 34, 35, 36, and 37.

Also, provided are fragments of at least 5 contiguous amino acidsequences corresponding to SEQ ID NOs: 2, 3, 4, 5, 6, and 7. Among thesefragments are those comprising PFVYG (SEQ ID NO: 32), NPQDW (SEQ ID NO:33), LPYGQ (SEQ ID NO: 34), IHRKR (SEQ ID NO: 35), LAVTI (SEQ ID NO:36), and NPFPG (SEQ ID NO: 37).

Methods of Synthesizing Polypeptides

The peptides, polypeptides, and polypeptide fragments disclosed hereincan be chemically synthesized using currently available laboratoryequipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City,Calif.). One skilled in the art can readily appreciate that a peptide orpolypeptide corresponding to the sodium channels disclosed herein, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide fragment can be synthesized and not cleavedfrom its synthesis resin whereas another peptide or polypeptide fragmentcan be synthesized and subsequently cleaved from the resin, therebyexposing a terminal group which is functionally blocked on the otherfragment. By peptide condensation reactions, these two fragments can becovalently joined via a peptide bond at their carboxyl and aminotermini, respectively, to form an sodium channel, or fragment thereof.(See Grant G A (1992) Synthetic Peptides: A User Guide. W.H. Freeman andCo., New York, N.Y. (1992); Bodansky M and Trost B., Ed. Principles ofPeptide Synthesis. Springer-Verlag Inc., New York, N.Y. (1993)).Alternatively, the peptide or polypeptide is independently synthesizedin vivo as described above.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen, etal. (1991) Biochemistry 30:4151). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson, et al.(1994) Science 266:776-779). The first step is the chemoselectivereaction of an unprotected synthetic peptide-alpha-thioester withanother unprotected peptide segment containing an amino-terminal Cysresidue to give a thioester-linked intermediate as the initial covalentproduct. Without a change in the reaction conditions, this intermediateundergoes spontaneous, rapid intramolecular reaction to form a nativepeptide bond at the ligation site. Application of this native chemicalligation method to the total synthesis of a protein molecule isillustrated by the preparation of human interleukin 8 (IL-8)(Baggiolini, et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis, et al.(1994) J Biol Chem 269:16075; Clark-Lewis, et al. (1991) Biochemistry30:3128; Rajarathnam, et al. (1994) Biochemistry 33:6623-30).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, et al. (1992)Science, 256:221). This technique has been used to synthesize analogs ofprotein domains as well as large amounts of relatively pure proteinswith full biological activity (deLisle Milton, et al. Techniques inProtein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).

Antibodies to Mutant Na_(v)1.7 Sodium Channels

The disclosed materials, compositions, articles, devices, and methodsdisclosed herein, in one aspect, relate to purified antibodies thatselectively bind to an epitope of a mutant Na_(v)1.7 sodium channelalpha-subunit. In one aspect, the purified antibody selectively binds toan epitope of the I62V mutant Na_(v)1.7 sodium channel alpha-subunit. Inanother aspect, the purified antibody selectively binds to an epitope ofthe P149Q mutant Na_(v)1.7 sodium channel alpha-subunit. In yet anotheraspect, the purified antibody selectively binds to an epitope of theN641Y mutant Na_(v)1.7 sodium channel alpha-subunit. In a furtheraspect, the purified antibody selectively binds to an epitope of theK655R mutant Na_(v)1.7 sodium channel alpha-subunit. In a still furtheraspect, the purified antibody selectively binds to an epitope of theI739V mutant Na_(v)1.7 sodium channel alpha-subunit. In one aspect, thepurified antibody selectively binds to an epitope of the L1123F mutantNa_(v)1.7 sodium channel alpha-subunit.

By “selectively binds” is meant that the antibody binds to the mutantNa_(v)1.7 sodium channel without appreciably binding to the non-mutantNa_(v)1.7 sodium channel. By “binding” is meant such that the signalthat indicates binding is at least about 1.5 times the signal for anon-binding control. Thus, without appreciable binding is meant lessthan or equal to 1.5 times the background of a non-binding control.

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies, as well as humanized, fully human,and non-human antibodies. Also provided are fragments of theseantibodies wherein the fragments selectively bind with epitopes ofmutant Na_(v)1.7 sodium channel alpha-subunits. The antibodies can betested for their desired binding activity using the in vitro assaysdescribed herein, or by analogous methods. Optionally, the antibodiesare labeled directly or indirectly and can be used with imagingtechnologies to detect expression of the mutant Na_(v)1.7.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison, et al. (1984) ProcNatl Acad Sci USA, 81:6851-6855).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler, et al. (1975) Nature 256:495). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the mutant Na_(v)1.7 channels described herein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding thedisclosed monoclonal antibodies can be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). Libraries of antibodies or activeantibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. Nos. 5,804,440 and6,096,441.

In vitro methods are also suitable for preparing monovalent antibodies,including, for example, scfv antibodies. Digestion of antibodies toproduce fragments thereof, particularly, Fab fragments, can beaccomplished using routine techniques known in the art. For instance,digestion can be performed using papain. Examples of papain digestionare described in WO 94/29348 and U.S. Pat. No. 4,342,566. Papaindigestion of antibodies typically produces two identical antigen bindingfragments, called Fab fragments, each with a single antigen bindingsite, and a residual Fc fragment. Pepsin treatment yields a fragmentthat has two antigen combining sites and is still capable of crosslinking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (See Zoller, (1992) J CurrOpin Biotechnol 3:348-354).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

Human Antibodies

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner, et al. (1991) JImmunol 147:86-95. Human antibodies (and fragments thereof) can also beproduced using phage display libraries (see Hoogenboom, et al. (1991) JMol Biol 227:381; Marks, et al. (1991) J Mol Biol 222:581).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits, et al. (1993)Proc Natl Acad Sci USA 90:2551- 2555; Jakobovits, et al. (1993) Nature362:255-258; Bruggermann, et al. (1993) Year in Immunol 7:33).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ line antibody gene array intosuch germ line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using the mutant Na_(v)1.7 sodium channels provided herein.

Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide regions of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (see Jones, et al. (1986)Nature 321:522-525; Reichmann, et al. (1988) Nature 332:323-327; Presta,(1992) Curr Opin Struct Biol 2:593-596).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones, et al. (1986) Nature321:522-525; Riechmann, et al. (1988) Nature 332:323-327; Verhoeyen, etal. (1988) Science 239:1534-1536), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. Nos. 4,816,567, 5,565,332, 5,721,367, 5,837,243, 5,939,598,6,130,364, and 6,180,377.

In one aspect, as a form of therapy, antibodies can be used toinactivate the function of a mutant protein.

Methods of Drug Screening and Delivery

The materials, compositions, articles, devices and methods disclosedherein, in one aspect, relate to a method of identifying a compound thatmodulates mutant Na_(v)1.7 sodium channels comprising contacting, with atest compound, a cell containing a mutant Na_(v)1.7 nucleic acid thatencodes a mutant Na_(v)1.7 sodium channel comprising one or moremutations at residue 62, residue 149, residue 641, residue 655, residue739, or residue 1123 of the channel; detecting Na_(v)1.7 sodium channelactivity; and comparing the Na_(v)1.7 sodium channel activity in thecontacted cell with the amount of Na_(v)1.7 sodium channel activity in acontrol cell, wherein the control cell is not contacted by the testcompound, an increased or decreased Na_(v)1.7 sodium channel activity inthe test cell as compared to the control cell indicating a compound thatmodulates mutant Na_(v)1.7 sodium channels. Detecting sodium channelactivity can be accomplished by methods known in the art. For example, asuitable protocol for detecting sodium channel activity is described inKausalia, et al. (2003) J. Neurophysiol. 10.1152/jn.00676.2003.

The cell can express the mutant channel naturally or can be geneticallymodified to do so. Optionally, the cell is an oocyte that expressed themutant sodium channel. The mutant sodium channel can be a I62V, P149Q,N641Y, K655R, I739V, or L1123F mutant. Optionally, a mutant channel cancomprise one or more of the site mutations.

Optionally, channel activity is tested using intracellular orextracellular recording to assess changes in membrane potentialassociated with sodium ion flux. Alternatively, imaging technologies canbe used to observe labeled ion flux. Expression can be assessed inXenopus oocytes or mammalian cells such as CHO, HEK and tsa201.Mutations may result in errors of protein trafficking and proteininteraction. As such, mutant channels can be assessed for their abilityto form functional channels in the cell membrane as opposed to beingretained in the endoplasmic reticulum by using labeled antibodies to thewild-type channel, or by attaching a common epitope to the channels andusing a specific antibody to that epitope. Mutations that alterinteractions with intracellular proteins, such as protein kinase A,protein kinase C or calmodulin kinase, or the sodium channelbeta-subunits, can be identified through yeast 2-hybrid studies,co-immunoprecipitation experiments or electrophysiological experiments.

Also, the materials, compositions, articles, devices, and methodsdisclosed herein, in one aspect, relate to a method of preventing orreducing the effects of neurologic disorders like febrile seizures,afebrile seizures, or epilepsy by treating a subject at risk forneurologic disorders with a composition that modulates mutant Na_(v)1.7levels. Thus, a subject with a mutation(s) in Na_(v)1.7 sodium channelalpha-subunits, consistent with a neurologic disorder or an increasedrisk of a neurologic disorder, can be treated with a compositioncomprising a mutant Na_(v)1.7 modulator identified or manufactured usingthe methods taught herein.

The materials and compositions disclosed herein can be administered invivo in a pharmaceutically acceptable carrier. By “pharmaceuticallyacceptable carrier” is meant a material that is not biologically orotherwise undesirable, i.e., the material may be administered to asubject, along with a modulator of Na_(v)1.7 sodium channel functionidentified or made by the methods taught herein, without causing anyundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the pharmaceutical composition inwhich it is contained. The carrier would naturally be selected tominimize any degradation of the active ingredient and to minimize anyadverse side effects in the subject, as would be well known to one ofskill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,although topical intranasal administration or administration by inhalantis typically preferred. As used herein, “topical intranasaladministration” means delivery of the compositions into the nose andnasal passages through one or both of the nares and can comprisedelivery by a spraying mechanism or droplet mechanism, or throughaerosolization of the composition. The latter may be effective when alarge number of animals are to be treated simultaneously. Administrationof the compositions by inhalant can be through the nose or mouth viadelivery by a spraying or droplet mechanism. Delivery can also bedirectly to any area of the respiratory system (e.g., lungs) viaintubation. The exact amount of the compositions required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the severity of the disorder being treated,the particular nucleic acid or modulator used, its mode ofadministration and the like. Thus, it is not possible to specify anexact amount for every composition. However, an appropriate amount canbe determined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands.

Liposomes are vesicles comprised of one or more concentrically orderedlipid bilayers which encapsulate an aqueous phase. They are normally notleaky, but can become leaky if a hole or pore occurs in the membrane, ifthe membrane is dissolved or degrades, or if the membrane temperature isincreased to the phase transition temperature. Current methods of drugdelivery via liposomes require that the liposome carrier ultimatelybecome permeable and release the encapsulated drug at the target site.This can be accomplished, for example, in a passive manner wherein theliposome bilayer degrades over time through the action of various agentsin the body. Every liposome composition will have a characteristichalf-life in the circulation or at other sites in the body and, thus, bycontrolling the half-life of the liposome composition, the rate at whichthe bilayer degrades can be somewhat regulated.

In contrast to passive drug release, active drug release involves usingan agent to induce a permeability change in the liposome vesicle.Liposome membranes can be constructed so that they become destabilizedwhen the environment becomes acidic near the liposome membrane (see,e.g., (1987) Proc Natl Acad Sci USA 84:7851; (1989) Biochemistry 28:908,which are hereby incorporated by reference in their entireties for theirteachings of liposome construction and administration). When liposomesare endocytosed by a target cell, for example, they can be routed toacidic endosomes which will destabilize the liposome and result in drugrelease.

Alternatively, the liposome membrane can be chemically modified suchthat an enzyme is placed as a coating on the membrane which slowlydestabilizes the liposome. Since control of drug release depends on theconcentration of enzyme initially placed in the membrane, there is noreal effective way to modulate or alter drug release to achieve “ondemand” drug delivery. The same problem exists for pH-sensitiveliposomes in that as soon as the liposome vesicle comes into contactwith a target cell, it will be engulfed and a drop in pH will lead todrug release. This liposome delivery system can also be made to target Bcells by incorporating into the liposome structure a ligand having anaffinity for B cell-specific receptors.

Compositions including the liposomes in a pharmaceutically acceptablecarrier are also contemplated.

Transdermal delivery devices have been employed for delivery of lowmolecular weight proteins by using lipid-based compositions (i.e., inthe form of a patch) in combination with sonophoresis. However, asreported in U.S. Pat. No. 6,041,253, which is hereby incorporated byreference in its entirety for the methods taught therein, transdermaldelivery can be further enhanced by the application of an electricfield, for example, by ionophoresis or electroporation. Using lowfrequency ultrasound which induces cavitation of the lipid layers of thestratum corneum, higher transdermal fluxes, rapid control of transdermalfluxes, and drug delivery at lower ultrasound intensities can beachieved. Still further enhancement can be obtained using a combinationof chemical enhancers and/or magnetic field along with the electricfield and ultrasound.

Implantable or injectable protein depot compositions can also beemployed, providing long-term delivery of the composition. For example,U.S. Pat. No. 6,331,311, which is hereby incorporated by reference inits entirety for protein depot compositions and uses, reports aninjectable depot gel composition which includes a biocompatible polymer,a solvent that dissolves the polymer and forms a viscous gel, and anemulsifying agent in the form of a dispersed droplet phase in theviscous gel. Upon injection, such a gel composition can provide arelatively continuous rate of dispersion of the agent to be delivered,thereby avoiding an initial burst of the agent to be delivered.

The test compound and modulator taught herein can be, but is not limitedto, antibodies, chemicals, small molecules, modified antisense RNAs,ions, siRNAs, receptor ligands, drugs and secreted proteins.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how theantibodies, polypeptides, nucleic acids, compositions, and/or methodsclaimed herein are made and evaluated, and are intended to be purelyexemplary of the invention and are not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers (e.g., amounts, temperature,etc.), but some errors and deviations should be accounted for.

Example 1

Febrile seizures are the most common seizure disorder of earlychildhood, exhibiting a prevalence of 2-5% in European and NorthAmerican children and as high as 9% in the Japanese. The incidence offebrile seizures in first-degree relatives is 31% (Aicardi, Epilepsy inChildren, Raven Press, New York, N.Y. (1994)), supporting a stronggenetic etiology of febrile seizures. The impact of febrile seizures isconsiderable because individuals who experience febrile seizures have a2-7% chance of developing afebrile seizures later in life (Annegers, etal. (1987) N Engl J Med 316:493-498). These later epileptic phenomenainclude cases of various generalized convulsive, as well as simple andcomplex partial seizures.

Linkage analysis of a febrile seizure kindred K4425 identified a 10 cMregion of no recombination on chromosome 2q24 (FEB3, OMIM 604403), whichcontains five sodium channel alpha-subunit genes (see Peiffer, et al.(1999) Ann Neurol 46:671-678). Three of the sodium channel genes withinthis critical region, Na_(v)1.1, Na_(v)1.2, and Na_(v)1.3, share over85% identity and are highly expressed in brain (see Catterall, (2000)Neuron 26:13-25). Na_(v)1.7, which also resides within this criticalgenetic interval, shares approximately 70-80% homology with Na_(v)1.1,Na_(v)1.2, and Na_(v)1.3 (see Catterall, (2000) Neuron 26:13-25;Sangameswaran, et al. (1997) J Biol Chem 272:14805-14809), is expressedprimarily in neurons of the dorsal root ganglia, and shows minimal to noexpression in brain (Felts, et al. (1997) Brain Res Mol Brain Res45:71-82; Toledo-Aral, et al. (1997) Proc Natl Acad Sci USA94:1527-1532). Consequently, Na_(v)1.7 has been classified as aperipheral nervous system channel (Catterall, (2000) Neuron 26:13-25;Goldin, et al. (2001) Annu Rev Physiol 63:871-894).

Recently, disease-causing mutations were identified in Na_(v)1.1 andNa_(v)1.2 in generalized epilepsy febrile seizure plus (GEFS+), afebrile seizure disorder that is subtly different from the phenotypedescribed in kindred K4425 (see Singh, et al. (1999) Ann Neurol45:75-81; Sugawara, et al. (2001) Proc Natl Acad Sci USA 98:6384-6389;Wallace, et al. (2001) Am J Hum Genet 68:859-865; Escayg, et al. (2000)Nat Genet 24: 343-345). Sequence analysis of an affected individual inK4425 did not yield any disease-causing variants in either of these twogenes, or in the closely related Na_(v)1.3 gene. Sequence analysis ofthe Na_(v)1.7 large intracellular loop between domains I and II revealeda missense change (N641Y) in all affected individuals of K4425 that wasabsent from 236 control chromosomes (see FIG. 2).

Na_(v)1.7 was then sequenced in a panel of 32 sporadic and familialcases with seizures occurring in the setting of a febrile illness andfive additional variants in Na_(v)1.7 that were not found in 180ethnically matched control chromosomes were identified (Table 2). Thesevariants were identified in the intracellular N-terminus (I62V), the DIS1-S2 extracellular loop (P149Q), the DI-DII intracellular loop (K655R),the DII S1 transmembrane domain (I739V), and the DII-DIII intracellularloop (L1123F) (see FIG. 3).

TABLE 2 Amino acid conservation and clinical findings associated withNav1.7 mutations Amino Acid Conservation Species Gene family FamilyClinical Findings Exon Mutation mou/rat/rabb Nav1.1/1.2/1.3 HistoryPresentation (age) Clinical Course 1 I62V √ √ — FS (2 yr) FS until 2 yr3 P149Q P/P/A √ — FS (2 yr) FS until 4 yr 11 N641Y √ V/A/T  +* FS (mean1.3 yr) GTC, PC, SP, GT, GA until 6- 16 yr 12 K655R √ R/R/R + FS (5 yr)IGE until 6 yr 13 I739V √ √ + FS (1 yr) IGE until 8 yr 17 L1123F √ A/A/L— epilepsy (5 mo) Intractable seizures Species: corresponding amino acidof Na_(v)1.7 of the mouse, rat and rabbit; Gene family: correspondingamino acid of Na_(v)1.1, Na_(v)1.2, and Na_(v)1.3; √, amino acid isidentical to human Na_(v)1.7. Family history: −, negative; +, positive.FS, febrile seizures; GTC, generalized tonic-clonic; PC, partialcomplex; SP, simple partial; GT, generalized tonic; GA, generalizedatonic; IGE, idiopathic generalized epilepsy; *family described in FIG.2.

All variants, except proline 149, are conserved in the Na_(v)1.7 gene ofmouse, rat and rabbit. Proline 149 is conserved in mouse and rat, and issubstituted with alanine in rabbit (Table 2). Less conservation of themutant Na_(v)1.7 residues is found among the Na_(v)1.1, Na_(v)1.2, andNa_(v)1.3 genes.

A broad variety of neurologic manifestations is observed in patientswith mutations in Na_(v)1.7, suggestive of a wide clinical continuum.Illustrating the milder end of the continuum are two probands sufferingonly of infrequent febrile seizures before six years of age (Table 2:I62V, P149Q). An additional two such patients later developed raregeneralized convulsive episodes (associated with generalizedepileptiform discharges on EEG) that resolved by eight years of age(Table 2: K655R, I739V). All 21 affected individuals in K4425experienced febrile seizures before six years of age (Table 2: N641Y).Eight of these individuals had later afebrile seizures which remitted bythe age of 16 in six individuals. Peiffer, et al. (1999) Ann Neurol46:671-678). These patients with afebrile seizures that ultimatelyresolved suggest an intermediate phenotype. Lastly, one proband in ourstudy experienced multiple generalized clonic seizures which werepredominantly afebrile, beginning at five months of age. This patientwithout a family history of seizures progressed to have frequentepisodes of status epilepticus and prolonged complex partial seizures by16 months, and at 5 years old, continues to have mixed seizures(including probable myoclonic and astatic seizures) in spite of resolutetherapeutic intervention. This last case represents the severe end ofthe clinical spectrum, and may be characterized as similar to SMEI(Table 2: L1123F). There is now abundant evidence for an increasingrange of epilepsy phenotypes in patients with mutations in Na_(v)1.1(see Nabbout, et al. (2003) Neurology 60:1961-1967; Fujiwara, et al.(2003) Brain 126:531-546). Electrophysiological characterization ofthese unique Na_(v)1.7 mutations may help shed light on the variation inseizure manifestation observed in this group of patients.

To date, Na_(v)1.1 and Na_(v)β1.1 are the most commonly mutated genes inthe febrile seizure phenotype. However, in an Australian cohort of 36unrelated GEFS+ samples, mutations in Nav1.1 and Na_(v)β1.1 account foronly 17% of cases (see Wallace, et al. (2001) Am J Hum Genet68:859-865). In our panel of 32 unrelated febrile seizure cases, onlyone Na_(v)1.1 mutation, R946H, was identified. Na_(v)1.1 is implicatedas a major cause of SMEI or Dravet syndrome (see Nabbout, et al. (2003)Neurology 60:1961-1967; Fujiwara, et al. (2003) Brain 126:531-546;Claes, et al. (2003) Hum Mutat 21:615-621). Since, GEFS+, and possiblySMEI, exhibit genetic heterogeneity, there can be a prevalence ofNa_(v)1.7 mutations in cohorts of both disorders.

Example 2

Experiments were conducted as described in Lossin, et al., (2003) JNeurosci 23(36):11289-11295. Results are shown in FIG. 4. Full-lengthwild-type SCN9A and mutant SCN9A (K655R and N641Y) constructs weretransiently transfected into tsA201 cells. Currents were elicted by testpulses from −60 mV to +40 mV in 5 mV increments. At negative potentials,K655R has a higher current density than wild type. At positivepotentials, N641Y has reduced current density compared to wild-type,p<0.05.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1-88. (canceled)
 89. A method for determining a subject's predispositionto a neurologic disorder associated with a sodium channel mutation,comprising the step of identifying mutations at one or more sites inregions of the nucleic acid sequence that encode an intracellularN-terminal region, an extracellular loop in domain I, an intracellularloop between domains I and II, an intracellular loop between domains IIand III, an intramembrane region of domain II, or any combinationthereof, such identified nucleotides indicating a predisposition to theneurologic disorder.
 90. The method of claim 89, wherein the mutation ispresent in the nucleic acid region encoding the intracellular N-terminusregion of the subunit.
 91. The method of claim 89, wherein the mutationis present in the nucleic acid region encoding the extracellular loop ofdomain I of the subunit.
 92. The method of claim 89, wherein themutation is present in the nucleic acid region encoding theintracellular loop between domains I and II of the subunit.
 93. Themethod of claim 89, wherein the mutation is present in the nucleic acidregion encoding the intracellular loop between domains II and III of thesubunit.
 94. The method of claim 89, wherein the mutation is present inthe nucleic acid region encoding the intramembrane region of domain IIof the subunit.
 95. The method of claim 89, wherein the step ofidentifying the mutations comprises comparing the nucleic acid sequenceto a wild-type nucleic acid sequence.
 96. The method of claim 95,wherein the wild-type nucleic acid sequence encodes the amino acidsequence of SEQ ID NO:
 38. 97. The method of claim 89, wherein theidentifying step comprises obtaining a biological sample and testing thesample to identify the nucleotides at the mutations sites of the nucleicacid contained therein.
 98. The method of claim 89, wherein theneurologic disorder is a seizure disorder.
 99. The method of claim 98,wherein the seizure disorder is a febrile seizure disorder.
 100. Amethod for determining a subject's predisposition to a neurologicdisorder associated with a sodium channel mutation comprising comparingthe subject's Nav1.7 haplotype with one or more reference haplotypesthat correlate with the neurologic disorder, a similar haplotype in thesubject's Nav1.7 haplotype as compared to the reference haplotype orhaplotypes indicating a predisposition to the neurologic disorder. 101.The method of claim 28, wherein the neurologic disorder is a seizuredisorder.
 102. The method of claim 29, wherein the seizure disorder is afebrile seizure disorder.